BDQ OFC Mockup - Gene-Quantification · 2017-06-26 · qPCR, dPCR & NGS 2017 8th International Gene...

Editor-in-Chief: Stephen Bustin

Managing Editors: Jim Huggett and

Justin O’Grady

ISSN 2214-7535 VOLUME 11S, 2017

qPCR, dPCR & NGS 2017

8th International Gene Quantifi cation Event

Symposium & Application Workshops & Industrial Exhibition

“Liquid Biopsy, Integrative Big Data Analysis, Biomarker Signature…and beyond”

3rd - 7th April 2017

TUM School of Live Sciences

Technical University of Munich, Freising-Weihenstephan, Germany

www.qPCR-dPCR-NGS-2017.net

Scientifi c Board Stephen Bustin, Mikael Kubista, Jim Huggett,

Jo Vandesompele, Justin O’Grady, Vladimir Benes, Michael W. Pfaffl

Scientifi c event coordination and editor of BDQ Supplement Issue 11S (2017)Michael W. Pfaffl

Contents lists available at ScienceDirect

Biomolecular Detection and Quantifi cation

journal homepage: www.elsevier.com/locate/bdq

Session LB1 – Liquid Biopsies & Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S1

Session LB2 – Liquid Biopsies & Molecular Dx 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S3

Session NGSDA – NGS Integrative Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S5

Session dPCR 1 – Digital PCR 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S7

Session LB3 – Liquid Biopsies & Molecular Dx 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S9

Session LTS-IDT – Technical Lunch-time Seminar by IDT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S11

Session LTS-Genex – Technical Lunch-time Seminar by MultiD & TATAA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S12

Session NGS – Next Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S12

Session ncRNA – non-coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S14

Session MIQE&QC – MIQE & qPCR Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S16

Session dPCR 2 – digital PCR 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S19

Session LTS-Kramer – Technical Lunch-time Seminar by Kramer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S21

Session DA – qPCR Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S21

Session MG – MicroGenomics & Single-Cell-qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S24

Postersessions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S25

Biomolecular Detection and Quantifi cation, 11S ( 2017 ) ii

CONTENTS

A

S

T

LW

S

SU

Eb

J

E

cwtw

h2

Biomolecular Detection and Quantification 11S (2017) S1–S53

Contents lists available at ScienceDirect

Biomolecular Detection and Quantification

journa l homepage: www.e lsev ier .com/ locate /bdq

bstracts - qPCR, dPCR & NGS 2017

8th international Gene Quantification Event

Symposium & Application Workshops & Industrial Exhibition

“Liquid Biopsy, Integrative Big Data Analysis, Biomarker Signature . . . and beyond”

3rd - 7th April 2017

TUM School of Live Sciences

Technical University of Munich, Freising-Weihenstephan, Germany

www.qPCR-dPCR-NGS-2017.net

Scientific board: Stephen Bustin, Mikael Kubista, Jim Huggett, Jo Vandesompele, Justin O’Grady,Vladimir Benes, Michael W. Pfaffl

Scientific event coordination and editor of BDQ Supplement Issue xxx (2017): Michael W. Pfaffl

ession LB1 – Liquid Biopsies & Exosomes

ime: Monday, 03/Apr/2017: 10:30 am – 12:30 pm

ocation: Lecture hall 14 – 650 participants – TUMeihenstephan

ession Chair: Michael W Pfaffl, TUM, Germany

ession Chair: Stephen Andrew Bustin, Anglia Ruskin University,nited Kingdom

xtracellular vesicles and their potential asiomarkers and therapeutics

an O. Lötvall

University of Gothenburg, Sweden-mail address: [email protected]

All cells in the body release multiple types of extracellular vesi-les (EVs), including exosomes, microvesicles and beyond. In 2007e discovered that exosomes contain both mRNA and microRNA

Different cells release EVs with different molecular cargos, andeach cell type can even change their EV cargo under differentconditions, such as stress or cancer transformation. This impor-tant feature of EVs has made them very interesting candidates asbiomarkers in disease. Indeed, both RNA and DNA EV cargo, aswell as EV proteins, can be utilized as diagnostics, and to monitordisease, which will be reviewed in the presentation.

The ability of EVs to shuttle RNA and other molecules fromone cell to the cytoplasm of another cell has also open up theopportunity to highjack this natural system to deliver therapeuticmolecules to diseased cells, where current biological treatmentssuch as antibodies are unable to reach. This opens up the oppor-tunity to develop treatments for different diseases, for exampletargeting oncogenes with microRNA or siRNA molecules, thatotherwise cannot easily penetrate the cell membrane. This newcategory of biological treatments has the opportunity to open upa totally new way to reach previously unreachable disease targets,with molecules that otherwise would not reach the ideal diseasetarget. This part of the presentation will thus describe the pos-

hat can be shuttled from one cell to another (Valadi et al., 2007),hich was a very surprising finding at the time.

ttp://dx.doi.org/10.1016/j.bdq.2017.02.001214-7535/

sibilities of EV-based therapeutics in different diseases, and give

dx.doi.org/10.1016/j.bdq.2017.02.001

http://www.sciencedirect.com/science/journal/00000000

www.elsevier.com/locate/bdq

http://www.qpcr-dpcr-ngs-2017.net/

http://crossmark.crossref.org/dialog/?doi=10.1016/j.bdq.2017.02.018&domain=pdf

mailto:[email protected]

dx.doi.org/10.1016/j.bdq.2017.02.001

S lar Det

ee

h

Ub

A

E

tHcsaSmcbctflteitaa

h

Df

V

E

cbafrs

ot[ssrar

atttp

[

[[

[

[

[

[

[

http://dx.doi.org/10.1016/j.bdq.2017.02.021

2 Abstracts qPCR dPCR & NGS 2017 / Biomolecu

xamples of models where this approach has been successful inxperimental models.

ttp://dx.doi.org/10.1016/j.bdq.2017.02.018

ltrasensitive mutation detection in liquidiopsies using SiMSen-Seq

nders Ståhlberg

University of Gothenburg, Sweden-mail address: [email protected]

Detection of cell-free DNA in liquid biopsies offers great poten-ial for use in non-invasive diagnostics and to monitor treatment.owever, the allele frequencies are often low in liquid biopsies andhallenging to analyze, since standard next-generation sequencinguffers from insufficient sensitivity and digital PCR is not suitable tonalyze multiple allele variants. Hence, we have developed SiMSen-eq: “Simple, Multiplexed, PCR-based barcoding of DNA for Sensitiveutation detection using Sequencing” that allows allele frequen-

ies <0.1% to be detected, using several kilobases of DNA. Severalarcoding strategies have been reported, but all require long andomplex library preparation protocols. SiMSen-Seq was developedo generate barcoded libraries with minimal DNA input (<5 ng),exible target selection and a very simple (∼3 h) library construc-ion protocol. SiMSen-Seq allows detection of variant alleles withasy customization of library content and a protocol that can bemplemented in any molecular biology laboratory. In this presen-ation we will discuss assay development and bioinformatics thatre used in SiMSen-Seq. In addition, we show data from severalpplications, focusing on cancer.


roplet-based digital PCR for cancer patientollow up

alerie Taly

Université Paris Descartes/INSERM/CNRS, France-mail address: [email protected]

Tumor-specific genomic alterations can be used to detect cir-ulating tumor DNA circulating (ctDNA). They represent specificiomarkers that distinguish cancer cells and normal cells. Theyre likely to be useful for the diagnosis, prognosis, treatment, andollow-up of cancer patients, but their use in clinical oncologyequires a highly sensitive strategy that discriminates modifiedequences in a large excess of DNA coming from normal cells [1,2].

Picoliter droplet-based digital PCR allows performing millionsf single molecule PCR reactions in parallel to detect and quan-ify a minority of mutant sequences in a complex mixture of DNA3]. It enables direct counting of the number of copies of a targetequences present in the sample and the multiplex detection ofeveral sequences including rare mutations and hypermethylatedegions [4,5]. Newly developed optimized NGS strategies can nowlso reach relevant sensitivity and allow the screening of a wideange of mutations in circulating DNA [6].

We will illustrate how, by allowing non invasive highly sensitivend quantitative analysis of ctDNA within blood patient samples,hese methods are used for follow-up of cancer progression andumor response to treatment. We will present results of prospec-ive clinical trials highlighting pertinence of these approaches in
ancreatic [7], lung [8] and colon cancer.
ection and Quantification 11S (2017) S1–S53

References

1] V. Taly, D. Pekin, A. El Abed, P. Laurent-Puig, Trends Mol. Med. 18 (7) (2012)405–416.

2] G. Perkins, H. Lu, F. Garlan, V. Taly, Adv. Clin. Chem. 79 (2017) (in press).3] D. Pekin, Y. Skhiri, J.-C. Baret, D. Le Corre, L. Mazutis, C. Ben Salem, F. Millot, A.

El Harrak, J.B. Hutchison, J.W. Larson, D.R. Link, P. Laurent-Puig, A.D. Griffiths,V. Taly, Lab Chip 11 (13) (2011) 2156–2166.

4] V. Taly, D. Pekin, L. Benhaim, S.K. Kotsopoulos, D. Le Corre, X. Li, I. Atochin, D.R.Link, A.D. Griffiths, K. Pallier, H. Blons, O. Bouche, B. Landi, J.B. Hutchison, P.Laurent-Puig, Clin. Chem. 59 (12) (2013) 1722–1731.

5] S. Garrigou, G. Perkins, F. Garlan, C. Normand, A. Didelot, D. Le Corre, S.Peyvandi, C. Mulot, R. Niarra, P. Aucouturier, G. Chatellier, P. Nizard, K.Perez-Toralla, E. Zonta, C. Charpy, A. Pujals, C. Barau, O. Bouché, J.-F. Emile, D.Pezet, F. Bibeau, J.B. Hutchison, D.R. Link, A. Zaanan, P. Laurent-Puig, I. Sobhani,V. Taly, Clin. Chem. 62 (8) (2016) 1129–1139.

6] N. Pecuchet, Y. Rozenholc, E. Zonta, D. Pietraz, A. Didelot, P. Combe, L. Gibault,J.B. Bachet, V. Taly, P. Laurent-Puig, H. Blons, E. Fabre, Clin. Chem. 62 (11)(2016) 1492–1503.

7] D. Pietraz, N. Pecuchet, F. Garlan, A. Didelot, O. Dubreuil, S. Doat, F.Imbert-Bismut, M. Karoui, J.C. Vaillant, V. Taly, P. Laurent-Puig, J.B. Bachet, Clin.Cancer Res. 23 (1) (2017) 116–123.

8] N. Pecuchet, E. Zonta, A. Didelot, P. Combe, C. Thibault, L. Gibault, Y. Rozenholc,V. Taly, E. Fabre, H. Blons, P. Laurent-Puig, PLoS Med. 13 (12) (2016) e1002199.


Exosomes – Advancing liquid biopsy diagnostics

Mikkel Noerholm

Exosome Diagnostics, GermanyE-mail address: [email protected]

The field of liquid biopsy has gained enormous interest in recentyears. An important goal of personalized medicine has been beingable to detect tumor derived genetic profiles and tracking the evo-lution of tumor mutations over time. Utilizing cell free tumor DNA(ctDNA) for detection of mutations in plasma has shown somepromise in late stage cancer patients, but can only be used to trackgenetic changes. A more complete picture can be seen by combin-ing the mutations in ctDNA with the mutations and RNA profilesfrom exosomes. Exosome Diagnostics have validated and launchedthe world’s first clinical tests using exosomal RNA (exoRNA). Wehave also developed a single step isolation platform for exoRNAand ctDNA from biofluids, which increases the available mutantcopies from the tumor available for analysis. In a blinded headto head analysis, this platform had better sensitivity when com-pared to ctDNA alone. Exosomes are especially interesting becausethey are released as an active process from not only cancer cells,but also other cells, such as tumor stroma and immune cells. Thisenables us to access RNA profiles when monitoring response tomore complex processes, such as immunotherapy response, wheremonitoring mutations may not be sufficient.

dx.doi.org/10.1016/j.bdq.2017.02.018



dx.doi.org/10.1016/j.bdq.2017.02.019



dx.doi.org/10.1016/j.bdq.2017.02.020



dx.doi.org/10.1016/j.bdq.2017.02.021

lar Det

S

T

LW

S

S

mb

J

E

pemtpatccmps

h

Sm

D

E

ibbdapoiRtprtim

SDamia

Abstracts qPCR dPCR & NGS 2017 / Biomolecu

ession LB2 – Liquid Biopsies & Molecular Dx 1

ime: Monday, 03/Apr/2017: 2:00 pm – 6:00 pm


ession Chair: Jo Vandesompele, Biogazelle, Belgium

ession Chair: An Hendrix, Ghent University, Belgium

RNA capture sequencing enabled liquidiopsy precision oncology

o Vandesompele

Biogazelle, Belgium-mail address: [email protected]

In contrast to general belief, a substantial part of the humanrotein coding transcriptome is abundantly present in the blood asxtracellular mRNA, ready to exploited. Here, I present probe basedRNA capture as a sensitive RNA sequencing workflow to study

housands of mRNA genes in cell-free RNA from cancer patients’lasma. Apart from RNA abundance profiling, this type of data canlso be use to detect structural RNA variants, such as somatic muta-ions, and RNA editing events, all known to play an important role inancer. RNA capture sequencing enables liquid biopsy guided pre-ision oncology, such as therapy stratification, treatment responseonitoring and early detection of relapse. I will also discuss the

reanalytical jungle of RNA targeted liquid biopsies and need fortandardization, as part of the ongoing exRNAQC study.


ensitive NGS method for the analysis oficrobial and viral nucleic acid in cell free RNA

oug Amorese

NuGEN Technologies, United States-mail address: [email protected]

Finding the causative agent of an infection can be challeng-ng. Low RNA quantity, poor quality, low viral titers, and highackground from the host negatively impact viral detection fromiofluids (blood, nasal swabs, CSF). PCR can be an effective means ofetecting specific, well characterized organisms but only if primersre closely spaced and those organisms are represented in the testanel. RNA-Seq is an alternative unbiased method that has previ-usly been used with cfRNA to identify unknown infections in anmpartial manner. However, this method typically requires higherNA input and quality than typically exist in clinical samples. Fur-her, the low titers of the pathogen RNA relative to the host and non-athogen RNA necessitate deep sequencing of the libraries. Severaleports have described the use of NuGEN’s isothermal amplificationechnology (SPIA) to overcome the low yields and poor RNA qual-ty for the detection of viruses in human biological fluids but these

ethods are still burdened with uninformative host transcripts.Here we describe a single workflow that combines NuGEN’s;

PIA amplification technology, enzymatic fragmentation, and Any-eplete (InDA-C targeted depletion method) to study the causative
gent(s) and host response in nasal samples obtained from asth-atic and non-asthmatic children with presumptive respiratory
nfections. This unbiased method enabled efficient viral detectionnd detection of lowly expressed human transcripts with <1 million

ection and Quantification 11S (2017) S1–S53 S3

reads per sample. Data illustrating the efficiency of the depletionmethod and the coverage of the viral genomes will be presented.


Identification of extracellular vesicle-specificbiomarkers

Jan Van Deun 1,3, Glenn Vergauwen 1,3,Pieter Mestdagh 2,3, Jo Vandesompele 2,3,Olivier De Wever 1,2, An Hendrix 1,2,∗

1 Laboratory of Experimental Cancer Research,Department of Radiation Oncology and ExperimentalCancer Research, Ghent University, Ghent, Belgium2 Department of Medical Genetics, Ghent University,Ghent, Belgium3 Cancer Research Institute Ghent, Ghent, BelgiumE-mail address: [email protected]

Extracellular vesicles (EVs) transmit information (nucleic acids,proteins and lipids) between different cell types, organs and evenorganisms, and have been detected in multiple body fluids. The con-nection of EVs to many aspects of human disease stimulated manyresearchers to explore their biomarker potential. The rapid expan-sion of the EV research field resulted in a struggle to cope with het-erogeneity in the implementation and reporting of isolation pro-tocols and characterization methods, delaying the introduction ofEV-specific biomarkers in clinical setting. We carried out a compar-ative study of commonly implemented EV isolation methods whichrevealed a method dependent outcome using omics approaches.Density gradient centrifugation minimized the co-isolation of(non)-membranous contaminants of different origin and obtaineda unique proteome and transcriptome signature. In addition, pre-analytical parameters that are commonly implemented but oftenvary among research groups, such as centrifugal filter types, wereshown to have an impact on EV analysis and should be care-fully considered and reported. To help overcome these issues inEV research, we established the EV-TRACK knowledgebase (www.evtrack.org), a crowdsourcing database that centralizes EV biologyand methodology. It currently comprises experimental parametersof over 1200 EV-related publications. The EV-TRACK platform aimsto stimulate authors, reviewers, editors and funders to put exper-imental guidelines into practice, which is a prerequisite to realizethe clinical potential of EV-related biomarkers.


NGS and qPCR analysis of RNA and microRNA inbiofluids and exosomes

Michael Thorsen

Exiqon, DenmarkE-mail address: [email protected]

Biofluids contain a multitude of RNA molecules some of whichmay serve as biomarkers for altered conditions in the body and beapplied in diagnosis of disease. Many biofluids are easily collectedand liquid biopsies hold the promise of developing non- or mini-mal invasive diagnostic tests for screening and monitoring diseases
such as cancer. Though rather unstable by nature, RNA in biofluidsor a fraction of these appear relatively stable, possibly because theyare protected from degradation within exosomes or within proteinaggregates.


dx.doi.org/10.1016/j.bdq.2017.02.022



dx.doi.org/10.1016/j.bdq.2017.02.023



http://www.evtrack.org/



dx.doi.org/10.1016/j.bdq.2017.02.024



S lar Det

RtscutaeLtapv

h

Rt

F

EF

isinegtvtsttb

h

Bch

W

E

yc

e

ra

tiiP



We are developing technologies and offer services to analyzeNA in biofluids which is challenged by the low content of RNA andhe presence of substances that interfere with the analyses. Exo-omes are of particular interest, as the RNA cargo that these vesiclesarry are derived from cells throughout the body and in partic-lar the cells in contact with the biofluid. Currently, we are ableo robustly analyze RNA including microRNA in different biofluidsnd exosomes using both next generation sequencing and LNATM

nhanced qPCR. Data will be presented showing how miRCURYNATM Universal RT microRNA PCR may be applied successfullyo validate NGS data. We will also share data on applying qPCRnalysis of urinary exosomes to discover microRNA biomarkers forrostate cancer and how a three-microRNA signature now has beenalidated in independent cohorts.


outine next-generation-sequencing of brainumors

elix Sahm

University Hospital Heidelberg, Germany-mail address:[email protected]

With the numbers of prognostic and predictive genetic markersn neuro-oncology steadily growing, the need for comprehen-ive molecular analysis of neuropathology samples has vastlyncreased. We developed an enrichment/hybrid capture-basedext-generation sequencing (NGS) gene panel comprising thentire coding and selected intronic and promoter regions of 130enes recurrently altered in brain tumors, allowing for the detec-ion of single nucleotide variations, fusions, and copy numberariations. Information derived from NGS data identified poten-ial targets for experimental therapy in about 75% of diagnosticamples. Such an approach will likely become highly valuable inhe near future for treatment decision making, as more therapeuticargets emerge and genetic information enters the classification ofrain tumors.


D Genomics: An integrated workflow for singleell analysis that helps to uncover tumoreterogeneity

ieland Keilholz

BD, Germany-mail address: [email protected]

Following the paradigm of easy-to-use tools for genomics anal-ses, BD Genomics offers a suite of products that focus on singleell applications.

The Tumor Dissociation reagent makes solid tumors from a vari-ty of tissues accessible to FACS technology.

The FACSMelodyTM is a benchtop cell sorter that enables inexpe-ienced FACS users to successfully run a sorting experiment throughutomation of complex tasks.

BD PreciseTM Assays are designed for transcriptome analysis on
he single cell level using an efficient workflow that seamlesslyntegrates with sorted cells. The assays ensure highest precisionn gene expression analysis by using molecular indexes to removeCR bias introduced during library preparation.

The assay is combined with access to efficient and widely auto-mated tools for the analysis of genomics data.

A case study will be presented on the integrated workflow oftumor dissociation, FACS sorting and transcriptome analysis. Newinsights into tumor biology at a currently unprecedented resolutionare revealed through this powerful approach.


Nucleosome association of cell-free DNAinforms about gene expression

Peter Ulz 1,∗, Gerhard Thallinger 2, Martina Auer 1,Ricarda Graf 1, Karl Kashofer 3,Stephan Wenzel Jahn 3, Luca Abete 3,Gunda Pristauz 4, Edgar Petru 4, Jochen Geigl 1,Ellen Heitzer 1, Michael Speicher 1

1 Institute of Human Genetics, Medical UniversityGraz, Austria2 Institute of Molecular Biotechnology, University ofTechnology, Graz, Austria3 Institute of Pathology, Medical University of Graz,Graz, Austria4 Department of Obstetrics and Gynecology, MedicalUniversity of Graz, Graz, AustriaE-mail address: [email protected]

Cell-free DNA (cfDNA) consists predominantly of nucleosome-protected DNA shed into the bloodstream by cells undergoingapoptosis. By whole-genome sequencing of cfDNA fragments tworegions which inform about nucleosome occupancy were identi-fied. Since nucleosome occupancy is a marker of gene expressionat transcription start sites (TSS), read depth differences may alsoinform about gene expression.

By a machine learning approach, gene expression was predictedfrom a pool of non-cancer controls and compared to previousgene expression results from studies of circulating RNA in healthydonors.

In tumor patients, this approach was applied to circulat-ing tumor DNA (ctDNA) whole-genome sequencing from regionsexhibiting copy-number alterations. Gene expression predictionwas compared to RNA-Seq from matching primary tumors andyielded high concordance.

Our analyses provide functional information about tumor cellsfrom DNA sequencing and adds an additional layer of informationto the analysis of ctDNA.

dx.doi.org/10.1016/j.bdq.2017.02.025



dx.doi.org/10.1016/j.bdq.2017.02.026



dx.doi.org/10.1016/j.bdq.2017.02.027



dx.doi.org/10.1016/j.bdq.2017.02.028

lar Det

S

T

LW

S

SU

Mmh

A

E

mtccavemsphtgose

h

Gt

T

1

2

E

nupBahtIsaINluaSs

[

[

[

those genes as well as a quantitative assessment. We consider thisapproach as “2nd generation full cycle” in microbial ecology.



ession NGSDA – NGS Integrative Data Analysis

ime: Monday, 03/Apr/2017: 2:00 pm – 6:00 pm


ession Chair: Vladimir Benes, EMBL, Germany

ession Chair: Daniel Simon Brewer, University of East Anglia,nited Kingdom

eta-OMIC reconstruction in the guticrobiome of wild primates: Impactions for

uman origins

ndres Gomez

J. Craig Venter Institute, United States-mail address: [email protected]

The concept of the holobiont considers both host and symbioticicrobes a single unit for selection in evolution. This idea suggest

hat the evolution process may not be fully understood withoutonsidering the role that residing microbes play in the physiologi-al landscape of the host. Here, I show that an extensive molecularnalysis of the primate gut microbiome offers a complementaryiew of the extrinsic and intrinsic forces that triggered humanvolution. To that end, I used integrated Meta-OMICS; mergingetagenomic, metabolomic and metatranscriptomics data from

tool samples to reconstruct the organizational and functional com-lexity of the gut microecosystem of wild gorillas, chimpanzees andumans. This comparative framework is implemented to assesshe potential impact that the gut microbiome has exerted in theenomic landscape of the host. In synthesis, these data sheds lightn how, over evolutionary timescales, diet and gut microbes inter-ected to influence energy harvest and immunity, impacting themergence of the lineage leading to humans.


ut microbiota sequencing: From single studieso large-scale analysis

homas Clavel 1,∗, Ilias Lagkouvardos 2

University Hospital RWTH Aachen, GermanyZIEL Institute, TU Munich, Freising, Germany-mail address: [email protected]

Since the discovery of Polymerase Chain Reaction and pio-eering work on 16S ribosomal RNA genes in the 1980s, these of molecular techniques to study microbial ecosystems andathogenic microorganisms has been increasing exponentially.ecause microbial communities inhabiting various body sites suchs the skin or the intestine can influence our physiology, thereas been a massive interest in studying their diversity, func-ions, and role in the development of acute and chronic diseases.n my presentation, I will talk about sequencing approaches totudy microbial communities, especially human body microbiota,nd give opinions on their potential and limitations. Moreover,will present a new bioinformatic tool, the Integrated Microbialext Generation Sequencing (IMNGS) platform, for user-friendly,

arge-scale studies of 16S rRNA gene amplicon datasets. IMNGSniformly and systematically screens for, retrieves, processes, and
nalyses all prokaryotic 16S rRNA amplicon data available in theequence Read Archive (SRA) and uses them to build sample-pecific sequence databases. Using a web interface, this massively

integrated sequence resource can be queried and used to addressa broad range of questions of relevance in microbial ecology andapplied microbiology. The platform also offers a complete work-flow for de novo analysis of users’ own raw 16S rRNA amplicondata. This new resource can be accessed at www.imngs.org [1–3].References

1] Clavel, Lagkouvardos, Hiergeist, Microbiome sequencing: challenges andopportunities for molecular medicine, Expert Rev. Mol. Diagn. 16 (2016) 795.

2] Lagkouvardos, Joseph, Kapfhammer, Giritli, Horn, Haller, Clavel, IMNGS: amassive open resource of processed 16S rRNA microbial profiles for ecologyand diversity studies, Sci. Rep. 6 (2016) 33721.

3] Lagkouvardos, Kumar, Fischer, Clavel, Rhea: a transparent and modular Rpipeline for microbial profiling based on 16S rRNA gene amplicons, PeerJ 5(2017) e2836.


From qualitative to quantitative data inmicrobiome analysis: Using metagenomics forqPCR validation

Michael Schloter 1,2,∗, Gisle Vestergaard 1,Anne Schoeler 1, Stefanie Schulz 1,Fabian Bergkemper 1

1 Helmholtz Zentrum München, Germany2 Technical University of Munich, GermanyE-mail address: [email protected]

Microbial diversity in nature is enormous and even today weare unable to predict the correct number of species which areharboured by most ecosystems including our human body. Thissituation becomes even worse when not one marker gene is stud-ied, like in the case of the analysis of species richness, but completemetagemones and the related functional traits should be described.Here also most next generation sequencing approaches fail assequencing efforts are needed to cover functional diversity whichgo far beyond 5 Tbases. Furthermore as still most of the func-tional traits are not well described and an annotation of readstowards predicted functions is hard to do or even impossible. Inthis presentation we describe an approach linking sequencing ofmetagenomes with a relative low coverage (10 Gbases or less), withthe aim to identify key genes of interest which drive certain pro-cesses and to use this information to develop highly targeted primerpairs, which might be useful for in depth analysis of the diversity of



dx.doi.org/10.1016/j.bdq.2017.02.029



http://www.imngs.org/



dx.doi.org/10.1016/j.bdq.2017.02.030



dx.doi.org/10.1016/j.bdq.2017.02.031

S lar Det

Ut

DCGC

1

U2

I3

CH4

K5

C6

H7

E

ecnd

tmmnacdcTcpcpEOh

lwnoloesscspadcttsti

as a response to these challenges. We designed our system fromscratch, to solve each of these problems in the best possible way.



nravelling the evolution of prostate cancerhrough whole genome sequence data

aniel S. Brewer 1,∗, David Wedge 2,3,hristopher S. Foster 4, David Neal 5,. Steve Bova 6, Andy G. Lynch 5,olin S. Cooper 7,1, Ros Eeles 7

Norwich Medical School, University of East Anglia,nited KingdomCancer Genome Project, Wellcome Trust Sanger

nstitute, United KingdomOxford Big Data Institute & Oxford Centre forancer Gene Research, Wellcome Trust Centre foruman Genetics, United KingdomHCA Pathology Laboratories, London, UnitedingdomCancer Research UK Cambridge Institute,ambridge, United KingdomUniversity of Tampere and Tampere Universityospital, FinlandInstitute of Cancer Research, United Kingdom-mail address: [email protected]

As part of the CRUK-ICGC Prostate UK project we have been gath-ring whole genome sequencing data from tissue from prostateancer patients and applying sophisticated data analysis tech-iques to unravel how prostate cancer evolves. In this talk we willescribe two projects:

Project 1 – Whole genome DNA sequencing was used to decrypthe phylogeny of multiple samples from distinct areas of cancer and

orphologically normal tissue taken from the prostates of threeen. Mutations were present at high levels in morphologically

ormal tissue distant from the cancer reflecting clonal expansions,nd the underlying mutational processes at work in morphologi-ally normal tissue were also at work in cancer. Our observationsemonstrate the existence of on-going abnormal mutational pro-esses, consistent with field-effects, underlying carcinogenesis.his mechanism gives rise to extensive branching evolution andancer clone mixing as exemplified by the coexistence of multi-le cancer lineages harbouring distinct ERG fusions within a singleancer nodule. Subsets of mutations were shared either by mor-hologically normal and malignant tissue or between differentRG-lineages, indicating earlier or separate clonal cell expansions.ur observations inform on the origin of multifocal disease andave implications for prostate cancer therapy in individual cases.

Project 2 – Cancers emerge from an on-going Darwinian evo-utionary process, often leading to multiple competing subclones

ithin a single primary tumour. This evolutionary process culmi-ates in the formation of metastases, which is the cause of 90%f cancer-related deaths. However, despite its clinical importance,ittle is known about the principles governing the disseminationf cancer cells to distant organs. Although the hypothesis thatach metastasis originates from a single tumour cell is generallyupported, recent studies using mouse models of cancer demon-trated the existence of polyclonal seeding from and inter-clonalooperation between multiple subclones. Using whole genomeequencing, we characterised multiple metastases arising fromrostate tumours in ten patients. Integrated analyses of subclonalrchitecture revealed the patterns of metastatic spread in unprece-ented detail. Metastasis-to-metastasis spread was found to beommon, either through de novo monoclonal seeding of daugh-er metastases or, in five cases, through the transfer of multiple
umour clones between metastatic sites. Lesions affecting tumouruppressor genes usually occur as single events, whereas muta-ions in genes involved in androgen receptor signalling commonlynvolve multiple, convergent events in different metastases. Our

results elucidate in detail the complex patterns of metastatic spreadand further our understanding of the development of resistance toandrogen deprivation therapy in prostate cancer.


Nanopore sequencing – Entering new next

Vladimir Benes ∗, Jonathan Landry,Jonathon Blake, Bettina Haase, Dinko Pavlinic,Jan Provaznik

EMBL, GermanyE-mail address: [email protected]

Advances in sequencing technologies have provided the scien-tific community with ample opportunities for almost unrestrictedexploration of organisms of their choice. However, complete denovo assembly of ‘larger’ genomes continues to be technically chal-lenging. Among other things, this barrier hinders genomics corefacilities from offering this application to their user base.

Ultimately, a straightforward system able to deliver compre-hensive information also on the primary code of organisms withuncharacterized genomes would ideally complement core facil-ities’ otherwise wide range of methods, which they utilize fordescription of other nucleic-acid cellular components.

EMBL GeneCore has applied nanopore sequencing for finish-ing several de novo genome sequencing projects. With simplifiedDNA isolation protocol enabling ‘reads’ over 100 kb long we havebeen able to considerably improve assembly of several 0.5–1 Gbgenomes. Positive results from our first attempts with new R9 flow-cells indicate that to achieve worthwhile genomes’ assemblies, wewill be able to reduce amount of data generated by short-readsequencing technology soon.


Overcoming NGS analysis bottlenecks with asystematic, scalable system

Amit Sinha

Basepair, United StatesE-mail address: [email protected]

Next generation DNA sequencing (NGS) has led to a paradigmshift in biomedical research. The ability to study the genome,transcriptome, epigenome, etc. has present many opportunities toaccelerate innovation in life sciences. While the cost and accessi-bility of NGS technology have improved exponentially, the biggestbottleneck is now data analysis and interpretation. NGS generatesseveral gigabytes of raw data for each sample, leading to hugecost for setting up storage and computation infrastructure. Theanalysis is complex, and requires installing and running severaldifferent software. Finally, communicating the research to physi-cians and scientists is cumbersome. Basepair has been developed



dx.doi.org/10.1016/j.bdq.2017.02.032



dx.doi.org/10.1016/j.bdq.2017.02.033



dx.doi.org/10.1016/j.bdq.2017.02.034

lar Det

Eh

C

E

tgtclmcecor

h

S

T

LW

SK

SL

U

J

E

yaa(aatrtoi

h


valuation of regulatory small variants inuman genetics

hristian Zinser

Genomatix GmbH, Germany-mail address: [email protected]

Next Generation Sequencing has become a basis for the detec-ion of small sequence variants in whole exomes and wholeenomes. The correct interpretation of the relevance of iden-ified variants is a major challenge in clinical diagnostics. Foroding single nucleotide variants, published standards and guide-ines have been widely adopted by clinical laboratories. The

ajority of disease-associated variants, however, are found in non-oding regions, suggesting that they affect the regulation of genexpression. Their analysis and reliable evaluation is methodicallyhallenging and so far not standardized. This talk will provide anverview of recent developments in the assessment of potentialegulatory variants.


ession dPCR 1 – Digital PCR 1

ime: Tuesday, 04/Apr/2017: 8:30 am – 12:30 pm


ession Chair: Jim Huggett, LGC & University of Surrey, Unitedingdom

ession Chair: Afif M. Abdel Nour, Holy Spirit University of Kaslik,ebanon (Lebanese Republic)

nprecedented accuracy when using digital PCR

im Huggett

LGC & University of Surrey, United Kingdom-mail address: [email protected]

Digital PCR (dPCR) offers a unique approach for molecular anal-sis as it counts DNA molecules, separated by limiting dilution, indefined volume. The method offers high quantitative precision

nd the fact that each DNA molecule has its own minute reactionor partition) means rare genetic variants are easier to detect. Thebility of dPCR to measure accurately (with both high precisionnd low bias) is of particular interest as it performs absolute quan-ification and thus may offer SI traceability. This could have wideanging implication for other methods such as qPCR and NGS. Thisalk will present the research into dPCR accuracy and discuss some
f the implications this may have on molecular measurement bothn research and more routine applications.


Liquid profiling of RNA and DNA for treatmentresponse prediction and monitoring of cancerpatients

Christof Winter

TU München, GermanyE-mail address: [email protected]

Cell-free nucleic acids released from dying tumor cells canreadily be detected in the blood plasma. To distinguish tumor fromnormal cell-free nucleic acids, tumor-specific mutations can beused. Here I will present the results of two studies using this liq-uid profiling approach for tumor load monitoring and personalizedtreatment in cancer patients.

In breast cancer, where the tumor mutation spectrum differssubstantially between patients, we make use of individual chro-mosomal rearrangements in the tumor that we detect and quantifyin patient blood over time using personalized droplet digital PCRassays. This allowed to retrospectively detect a metastatic recur-rence in blood on average 11 months before clinical detectionwith symptoms. Early detection and early treatment might leadto improved patient survival.

In prostate cancer, the androgen receptor (AR) is an importanttarget for hormone therapy (androgen deprivation therapy). How-ever, a considerable fraction of tumors expresses a splice variant(AR-V7) that is resistant to hormone therapy. We designed dropletdigital PCR assays specific for AR and for AR-V7 and then quan-tified RNA levels of these transcripts in patient whole blood. Inpatients with metastatic prostate cancer, we found that high AR-V7 blood levels before therapy initiation were associated with poorprogression-free survival and poor overall survival under hormonetherapy. Notably, we found that none of the patients with highAR-V7 levels did respond to a subsequent hormone therapy. Trans-lating these results into routine clinical testing will contribute toprecision oncology.


RT-Digital PCR for the evaluation of residualdisease in chronic myeloid leukaemia

Mary Alikian 1,2,∗, Olga Tatarinova 1,Alistair Reid 1, Jane Apperley 2,3, Letizia Foroni 2,3

1 Imperial Molecular Pathology, Imperial HealthcareTrust, Hammersmith Hospital, London, UK2 Centre for Haematology, Faculty of Medicine,Imperial College London, London, UK3 Clinical Haematology, Imperial College HealthcareNHS Trust, London, UKE-mail address: [email protected]

Tyrosine Kinase Inhibitors (TKIs) are part of the successful clin-ical management of patients with Chronic Myeloid Leukaemia(CML). However, optimal clinical management of CML requires arobust, standardised laboratory assay used at key clinical mile-stones to ensure a successful outcome for patients on TKIs.Quantitative monitoring of %BCR-ABL1IS by reverse transcriptionquantitative PCR (RT-qPCR) is the gold standard strategy for evalu-ating patient response to therapy and classification into prognosticsubgroups. However, it can be challenging to perform in a repro-ducible manner. Reverse-Transcription Digital PCR (RT-dPCR) is anadaptation of this method that could provide the robust and stan-
dardised workflow needed for patient stratification.
Recent research from our lab showed that RT-dPCR, using theEurope Against Cancer (EAC) assay, did not significantly improvethe sensitivity of residual disease quantification below Major



dx.doi.org/10.1016/j.bdq.2017.02.035



dx.doi.org/10.1016/j.bdq.2017.02.036



dx.doi.org/10.1016/j.bdq.2017.02.037



S lar Det

Moatct

tTqpwUicqnss

oc

h

CD

ERJEJ

1

U2

I3

LUL4

G5

&G6

NE

iStcmvoaf8poam


olecular Response (MMR). The presence of a considerable amountf background noise in the negative samples did not allow forn increase in the sensitivity. However, using RT-dPCR simplifiedhe quantification process eliminating the need for using standardurves and enabling the absolute quantification of the BCR-ABL1ranscript molecules expressed as transcript copies per reaction.

In this study, we validated a new assay, called IDT, for the quan-ification of BCR-ABL1 transcripts on the RainDropTM dPCR platform.he IDT assay uses GUSB as a reference gene and is capable ofuantifying the e13a2 and e14a2 transcript types of BCR-ABL1 inde-endently in each sample in a multiplex format. Using this assay,e quantified MRD in 138 samples from 64 patients enrolled in theK based De- Escalation and Stopping Treatment of Imatinib, Nilot-

nib or sprYcel in Chronic Myeloid Leukaemia (DESTINY) trial andompared the BCR-ABL1 transcripts numbers quantified by both RT-PCR and RT-dPCR. Although the IDT assay had significantly loweroise levels, the assay did not make a significant difference on theensitivity compared to RT-qPCR. This result alludes to the fact thatamples in deep molecular response are truly disease free.

In this work, we introduce the IDT assay coupled with RT-dPCRn the RainDrop platform for the quantification of BCR-ABL1 trans-ripts for monitoring MRD in CML patients.


alibrating quantitative measurements of HIVNA using digital PCR

loise Busby 1,∗, Alexandra Whale 1,. Bridget Ferns 2, Paul R. Grant 3, Gary Morley 1,

onathan Campbell 1, Carole Foy 1,leni Nastouli 3,4, Jim Huggett 1,5,eremy A. Garson 2,6

Molecular and Cell Biology Team, LGC, Teddington,KDepartment of Infection, Division of Infection and

mmunity, University College London, London, UKDepartment of Clinical Virology, University Collegeondon Hospital NHS Foundation Trust, and theCL/UCLH NIHR Biomedical Research Centre,ondon, UKDepartment of Population Policy and Practice, UCLOS Institute of Child Health, London, UKSchool of Biosciences & Medicine, Faculty of HealthMedical Science, University of Surrey, Guildford

U2 7XH, UKNational Transfusion Microbiology Laboratories,HS Blood and Transplant, Colindale, London, UK-mail address: [email protected]

Quantification of HIV DNA associated with the viral reservoirs increasingly used in research as a tool to study latent disease.uch a target could potentially be used clinically to assist moni-oring of disease progression or decline as it has been reported toorrelate with viral outgrowth, and could serve as a biomarker foronitoring chronic infection. In addition to measurement of RNA

iral load, qPCR is used as the method of choice for quantificationf HIV DNA. Results are normalised to a human genomic targetnd reported as HIV DNA copies per 1,000,000 cells. When per-orming qPCR this is commonly achieved by calibrating against theE5 cell line reported to contain one stably integrated HIV genomeer cell. In this study we used dPCR to investigate the stability
f the HIV genome in the 8E5 cell line and demonstrated howccurate value assignment of this calibrator impacts upon qPCReasurement of HIV DNA. DNA from three separate sources of 8E5

cells, which included serially passaged fresh cells, was analysedusing two droplet-based dPCR platforms. We identified geneticinstability in the 8E5 cell line, which supports very recent find-ings of other researchers, and resulted in HIV DNA quantificationin patient samples varying by a factor of ∼50. dPCR value assign-ment of the calibrators removed this error. Appropriately calibratedquantitative methods afford greater accuracy and measurementharmonisation when using qPCR to measure a specific sequence.We show here that dPCR is a method that can effectively be usedto quantify calibration materials for this purpose.


Monitoring EGFR mutations in lung cancerpatients using 3-color Crystal Digital PCR

Rémi Dangla

Stilla Technologies, FranceE-mail address: [email protected]

Presentation of the Naica System, a fast and user-friendly digitalPCR system uniquely equipped with a 3-color readout capabilityfor straightforward multiplexing. Application of the Naica Systemto monitor the treatment response of lung cancer patients fromctDNA, using a single 3-plex assay targeting both the sensitizing andresistance mutations. Preview on upcoming extended multiplexingcapacity.


Transferring qPCR methods into a dropletdigital PCR format – Experiences from OfficialFood and Feed Control in GMO analysis

Sven Pecoraro

Bavarian Health and Food Safety Authority, GermanyE-mail address: [email protected]

According to Regulation (EC) No 1829/2003 genetically modi-fied organisms (GMO) have to be approved to be put on the marketin the EU. Food or feed that contain, consist or are produced fromGMO have to be properly labelled. This provision shall not apply upto a proportion of 0.9% of a given GMO material, provided that thepresence of such material is adventitious or technically unavoid-able. From this it follows for official food and feed control, that ifGMO material is qualitatively detected in a given sample the pro-portion of this material (taxon related) has to be quantified withqPCR. Quantification in GMO analysis consists of two independentqPCR reactions. In one reaction the amount of taxon (plant) spe-cific DNA is measured relative to a standard curve with knownDNA copy numbers. The other reaction measures the DNA copynumbers of the specific GMO. These two DNA copy numbers aredivided (cp gmo/cp reference gene) and multiplied by 100% to givethe final GMO percentage (cp/cp). Typically the limit of quantifica-tion (LOQ) of qPCR in GMO analysis is 0.1%. According to Regulation(EU) No 619/2011 certain GMOs have to be accurately quantified ata GMO proportion of 0.1%. However, quantifying at the lower endof the dynamic range of qPCR can be inaccurate. Digital droplet PCR(ddPCR) offers the advantage that DNA copy numbers can be abso-lutely quantified without the need for standard curves. AdditionallyddPCR is capable to accurately measure minor quantities of DNA.Furthermore ddPCR is considered as less susceptible to inhibitory
effects which is relevant when analyzing DNA that derives fromcomplex matrices like food or feed. Official control laboratorieshave to apply validated methods. In GMO analysis predominantly
dx.doi.org/10.1016/j.bdq.2017.02.038



dx.doi.org/10.1016/j.bdq.2017.02.039



dx.doi.org/10.1016/j.bdq.2017.02.040



lar Det

mWsibeprttaarpsHiaw

h

Sn

P

BE

smtmpabftrsnnnsapsmdcn

C

h


ethods published by the Joint Research Centre (JRC) are used.hen such qPCR methods are transferred into a ddPCR format

everal aspects should be considered. As usually the master mixs different in ddPCR than for qPCR, a selected set of GMO shoulde tested in order to assess the specificity of the reaction. In gen-ral the reaction conditions like annealing temperature, primer androbe concentrations should be taken as they were validated. Ifesults are not satisfying, e.g. because the separation between posi-ive and negative signals is not optimal (intermediate signals = rain)hen a temperature gradient can be performed to evaluate the mostppropriate annealing temperature. One has to consider that if thennealing temperature is altered more than 1 ◦C to 2 ◦C (robustnessange) then the method has possibly to be re-validated. Increasedrimer and/or probe concentrations can also lead to better signaleparation. The experience of the GMO laboratory of the Bavarianealth and Food Safety Authority (LGL) with ddPCR is very promis-

ng. The procedure is accurate even with minor quantities of DNAnd serves as an alternative reference method to standard qPCRhen quantifying GMO DNA derived from complex matrices.


imultaneous quantification of DNA copyumber and transcripts by Selfie-dPCR

etar Podlesniy ∗, Ramon Trullas

Institute of Biomedical Research of Barcelona,arcelona, Spain-mail address: [email protected]

To understand the mechanisms that regulate DNA gene tran-cription and replication it is necessary to have a preciseeasurement of the number of transcripts per gene. However, in

he case of mitochondrial DNA (mtDNA), precise measurement oftDNA transcription requires to take into account that mtDNA is

resent in a number of copies that varies depending on cell typend conditions, it is expressed in a polycistronic transcript andoth light and heavy chains express their own transcript in a dif-erentially regulated way. The presence of two mtDNA encodedranscripts that are regulated independently prevents their use aseference transcripts. In addition, distinction between the expres-ion of the transcripts of the light and heavy chain is usuallyot achieved in the standard PCR workflow. Here we present aovel method named Selfie-PCR that allows the precise simulta-eous measurement of both genomic and mtDNA transcripts in theame sample. The number of transcripts per encoding gene can bessessed in a locus specific and strand specific manner. Selfie-PCRermits the quantification of transcription initiation events in bothtrands and the assessment of gene transcription progress. As thisethod uses genes present in the sample as the own reference stan-

ards and does not rely on any external reference it can be used inells and tissues from different origins with different gene copyumber or metabolic state.

Acknowledgement: Supported by SAF2014-56644-R andIBERNED PI2016/06-3 grants.



Session LB3 – Liquid Biopsies & Molecular Dx 2

Time: Tuesday, 04/Apr/2017: 8:30 am – 12:30 pm

Location: Lecture hall 15 – 350 participants – TUMWeihenstephan

Session Chair: Kenneth W Witwer, Johns Hopkins UniversitySchool of Medicine, USA

Session Chair: Michael W Pfaffl, TUM, Germany

Extracellular vesicle subsets and samplingstrategies for nucleic acid profiling

Kenneth W. Witwer

Johns Hopkins University School of Medicine, UnitedStatesE-mail address: [email protected]

Enthusiasm continues unabated for the clinical potential ofextracellular RNA biomarkers, especially those associated withextracellular vesicles (EVs) that may be traced back to a cell of ori-gin. At the same time, it has become apparent that a diversity of EVsare produced by cells under normal and pathological conditions.In this presentation, data from three “challenging biofluid studies”are shared to demonstrate the influence of sampling strategy onEV subsetting and nucleic acid profiling results. Specifically, samp-ling site (for blood) is shown to affect recovery of large EVs; bloodadditives substantially influence small RNA profiling results; anddifferent sampling strategies for cervicovaginal secretions affectinterpretation of small RNA content by qPCR and qPCR array.


Cellular and extracellular miRNAs are bloodcompartment-specific diagnostic targets fordetection and survival prediction in sepsis

Marlene Reithmair 1,∗, Dominik Buschmann 1,2,Melanie Märte 3, Benedikt Kirchner 2,Daniel Hagl 3,4, Ines Kaufmann 4, Martina Pfob 1,Alexander Chouker 3, Ortrud Steinlein 1,Michael W. Pfaffl 2, Gustav Schelling 3

1 Institute of Human Genetics, University Hospital,Ludwig-Maximilians-University, Munich, Germany2 Division of Animal Physiology and Immunology,TUM School of Life Sciences Weihenstephan,Technical University Munich, Germany3 Department of Anesthesiology, University Hospital,Ludwig-Maximilians-University, Munich, Germany4 Department of Anaesthesiology, NeuperlachHospital, City Hospitals of Munich, Munich, GermanyE-mail address:[email protected]

Background: Septic shock is a common medical condition witha mortality approaching 50% where early diagnosis and treatmentis of particular importance for patient survival. Novel biomark-ers that serve as prompt indicators of sepsis are urgently needed.High-throughput technologies assessing circulating microRNAsrepresent an important tool for biomarker identification but theblood-compartment specificity of these miRNAs has not yet been
investigated.
Methods: We characterized miRNA profiles from serum exo-somes, total serum and blood cells (leukocytes, erythrocytes,

dx.doi.org/10.1016/j.bdq.2017.02.041



dx.doi.org/10.1016/j.bdq.2017.02.042



dx.doi.org/10.1016/j.bdq.2017.02.043



S lar Det

pibtr

dlhtpimswca

cnpo

h

Dmfi

AKA

1

M2

E

scisctrvo

t9aaC0woPTpmf

h


latelets) of sepsis patients (n = 22) by next-generation sequenc-ng and RT-qPCR and established differences in miRNA expressionetween blood compartments. In-silico analysis was used to iden-ify compartment-specific signaling functions of differentiallyegulated miRNAs in sepsis-relevant pathways.

Results: In septic shock, a total of 77 and 103 miRNAs wereown- and upregulated, respectively. A majority of these regu-

ated miRNAs (14 in serum, 32 in exosomes, 73 in blood cells)ad not been previously associated with sepsis. We found a dis-inctly compartment-specific regulation of miRNAs between sepsisatients and healthy volunteers. Blood cellular miR-199b-5p was

dentified as a potential early indicator for sepsis and septic shock.iR-125b-5p and miR-26b-5p were uniquely regulated in exo-

omes and serum, respectively, while one miRNA (miR-27b-3p)as present in all three compartments. In-silico analysis identified

rucial signaling pathways such as LPS-stimulated toll-like receptornd HIF-1a-signaling regulated by exosome derived miRNAs.

Conclusions: The expression of sepsis-associated miRNAs isompartment-specific. Exosome-derived miRNAs contribute sig-ificant information regarding sepsis diagnosis and survivalrediction and could serve as newly-identified targets for the devel-pment of novel sepsis biomarkers.


iagnostic performance of plasma DNAethylation profiles in lung cancer, pulmonary

brosis and COPD

ndreas Weinhäusel 1,∗, Matthias Wielscher 1,lemens Vierlinger 1, Rolf Ziesche 2,ndrea Gsur 2, Christa Noehammer 1

AIT Austrian Institute of Technology GmbH,olekulare Diagnostik, AustriaMedical University Vienna, Austria-mail address: [email protected]

Disease-specific alterations of the cell-free DNA methylationtatus are frequently found in serum samples and are currentlyonsidered to be suitable biomarkers. Candidate markers weredentified by bisulfite conversion-based genome-wide methylationcreening of lung tissue from lung cancer, fibrotic ILD, and COPD.fDNA from 400 �l serum (n = 204) served to test the diagnos-ic performance of these markers. Following methylation-sensitiveestriction enzyme digestion and enrichment of methylated DNAia targeted amplification (multiplexed MSRE enrichment), a totalf 96 markers were addressed by highly parallel qPCR.

Lung cancer was efficiently separated from non-cancer and con-rols with a sensitivity of 87.8%, (95%CI: 0.67–0.97) and specificity0.2%, (95%CI: 0.65–0.98). Cancer was distinguished from ILD withspecificity of 88%, (95%CI: 0.57–1), and COPD from cancer withspecificity of 88% (95%CI: 0.64–0.97). Separation of ILD from

OPD and controls was possible with a sensitivity of 63.1% (95%CI:.4–0.78) and a specificity of 70% (95%CI: 0.54–0.81). The resultsere confirmed using an independent sample set (n = 46) by use

f the four top markers discovered in the study (HOXD10, PAX9,TPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72–0.95).his technique was capable of distinguishing interrelated complexulmonary diseases suggesting that multiplexed MSRE enrichment
ight be useful for simple and reliable diagnosis of diverse multi-
actorial disease states.



HP’s advanced microfluidic technology

Alexander N. Govyadinov

HP Incorporated, United StatesE-mail address: [email protected]

Recently, there is a lot of interest in microfluidic lab-on-a chip application for life science, forensic, point-of-care,molecular-diagnostic, other in-vitro-diagnostic, organs-on-a-chip,environmental and multiple others applications. Different scien-tific and commercial organizations explore a multitude of materialsets and operational principles to forge microfluidic devices. Simul-taneously, inkjet industry utilizes well established materials andprinciples of operation for complicated microfluidic systems devel-oped for precision dispensing and manipulation of droplets withpico-liter accuracy on a massively parallel scale. The presen-tation describes our recent progress of low cost microfluidicplatform development utilizing materials and processes developedfor low-cost thermal inkjet business. The concept repurposes wellestablished inkjet processes, microfluidic components and jettingelements for pumping, mixing, valving, fluid transport, sensing andother critical functions of complex integrated microfluidic systems.This presentation describes operating principles of microfluidicelements, examples of their integration in functional devices anddiscusses inkjet technology potential for broad range of microflu-idic applications.


NGS analysis of the liquid biopsy in metastaticbreast cancer patients: A pilot study

Pamela Pinzani ∗, Francesca Salvianti,Mario Pazzagli

University of Florence, ItalyE-mail address: [email protected]

Circulating tumor cells (CTCs) and cell-free DNA (cfDNA),released in the bloodstream from primary tumor and metastases,are considered a “liquid biopsy” of the tumor reflecting diseasecomplexity and evolution at any stage.

In a previous study we demonstrated the feasibility of a proto-col for the mutational analysis at the single cell level in patientsaffected by metastatic breast cancer.

The aim of the present study is the optimization of a protocolfor the detection of somatic mutations in cfDNA by Next Genera-tion Sequencing (NGS) and the comparison with data obtained fromsingle circulating tumor cells isolated from the same blood draw.Notwithstanding that the two components of the liquid biopsy,CTCs and cfDNA, have been extensively studied separately, fewstudies compared the information obtained from these two differ-ent compartments for a personalized therapeutic approach.

Cell-free DNA was extracted from 2 ml plasma by using theQIAamp circulating nucleic acid kit (QIAgen). DNA samples weresequenced by Ion s5 Sequencer by using the ion AmpliSeq Can-cer Hotspot Panel v2 (Thermofisher Scientific) targeting hotspotregions in 50 oncogenes and tumor suppressor genes which arefrequently mutated in cancer patients.

Results obtained from cfDNA of 5 metastatic breast cancerpatients have been compared with those of 3–5 CTCs isolated fromthe same blood draw and the primary tumor.

From the comparison we evidenced the presence of some con-
cordant mutations between tissue and liquid biopsy, but alsodiscrepancies.
In at least one patient no common somatic mutation was foundbetween tissue and liquid biopsy. In one subject two hotspot

dx.doi.org/10.1016/j.bdq.2017.02.044



dx.doi.org/10.1016/j.bdq.2017.02.045



dx.doi.org/10.1016/j.bdq.2017.02.046



lar Det

mnt

tpic

h

Vk

A

E

qsAIfpoeRk

h

G

O

E

dtstti

sci

mmipP

gctc

h


utations in TP53 were evidenced in CTCs and primary tumor butot in cfDNA while another mutation in the same gene was commono the tissue and cfDNA but could not be found in the CTCs.

This preliminary study suggests that CTCs and cfDNA representwo complementary aspects of the liquid biopsy. Our results sup-ort the feasibility of the use of the liquid biopsy as a useful tool

n personalized medicine for the management of metastatic breastancer patients.


alidation of clinical diagnostic real-time PCRits on AriaMX-DX

nna Gani ∗, Silvia Mason

AB ANALITICA, Italy-mail address: [email protected]

Molecular diagnostic laboratories are requesting more fre-uently fully automated solutions to obtain the maximum level oftandardization, reliability, traceability and efficiency. Recently, ABNALITICA’s bioengineering team has developed the GENEQUAL-

TY X120, a fully-integrated platform, for extraction of nucleic acidsrom a wide range of clinical samples and automated Real-Time PCRlate setup. A key point of the analytic process remains the choicef the Real-Time PCR instrument. In this study, we have started thevaluation of Agilent AriaMX-DX instrument with AB ANALITICAEALQUALITY kits and clinical validation of a new HPV screeningit.


oing to the limits of multiplex qPCR

lfert Landt

TIB MOLBIOL, Berlin, Germany-mail address: [email protected]

Disease with the same symptoms can be caused by up to twoozens different pathogens. Successive single pathogen testing isoo slow; parallel testing costs too many wells and is too expen-ive. Multiplex PCR testing increases the throughput and reduceshe costs per assay and target while providing at least a semiquan-itative result, helping to rate the significance of the results and todentify putative contaminations.

Some pathogens can cause different diseases. We combineingle target assay modules in one multiplex reaction allowingustomized combinations for multiplex testing using Roche 480nstruments.

Multiplex realtime qPCR is limited by the number of instru-ent dye channels but also by molecular interferences. Highlyultiplexed PCR assays are therefore limited in their sensitivity,

n particular if run as 1-step RT PCR on RNA. Stategies to weakenrimer binding during the reverse transcription step increases theCR perfomance.

For testing on bacterial resitance we combine routinely 10 tar-ets in one well, reaching a sensitivity of significant less than 10opies per reaction. In the Respiratory Viral Panel we combine rou-inely 11 targets in one well, achieving for the most targets still 10opies per reaction.



Session LTS-IDT – Technical Lunch-time Seminar byIDT

Time: Tuesday, 04/Apr/2017: 1:00 pm–2:00 pm

Location: Lecture hall 14, 650 participants – TUM Weihenstephan

Session Chair: Caifu Chen, Integrated DNA Technologies, UnitedStates of America

Novel multiplex rhPCR and its applications inamplicon sequencing

Caifu Chen

Integrated DNA Technologies, USAE-mail address: [email protected]

We report here a novel multiplex PCR chemistry called rhPCR. Itsprimers contain a non-extendable terminal blocker and single RNAbase closer to 3′. rhPCR requires both thermostable Type II RNase H(RNase H2) and DNA Polymerase for specific amplification. RNaseH2 can activate rhPCR primers by target-specific cleavage of theRNA base while rhPCR primers perfectly bind to the target DNA toform a DNA duplex. Cleaved rhPCR primers can then be extended bya DNA polymerase. Combination of both 3′ blocked rhPCR primersand a highly specific mutant DNA polymerase have eliminatedor greatly reduced primer dimers and non-specific amplification,resulting in higher multiplexity and better specificity than standardmultiplex PCR. We have demonstrated the feasibility of the multi-plex rhPCR for amplicon sequencing in multiple assay pools rangingfrom 96- to 1,000-plexes. Major advantages of multiplex rhPCRfor amplicon sequencing include better workflow, higher map-pable reads (>97%) and on-target percentage (>97%) comparing tostandard multiplex PCR.


rhAmpTM a better and cost-effective new SNPgenotyping solution

Kristin Beltz

Integrated DNA Technologies, Inc., USAE-mail address: [email protected]

rhAmpTM SNP Genotyping is a novel target-activated geno-typing solution utilizing RNase H2-dependent PCR (rhPCR) toprovide superior discrimination of single nucleotide polymor-phisms (SNPs), multi-nucleotide polymorphisms (MNPs), andinsertion/deletions (InDels). rhPCR genotyping technology com-bines a unique two enzyme system with DNA-RNA hybrid primersto interrogate target SNPs. Allele specific primers contain a 5′ uni-versal tail, a single RNA base targeting the SNP, and a terminalblocking group. The 3′ blocking group is removed and primer acti-vation is achieved only upon hybridization to its perfectly matchedtarget. The thermostable RNase H2 cleaves the primer at the RNAbase and releases the blocking group, allowing primer extension.PCR specificity and selectivity is improved with the use of a mutantTaq DNA polymerase and signal generation is achieved using a cost-effective universal reporter system. More than 550 human targetswere selected for testing. A proprietary algorithm provided a designrate greater than 95%, and consistently high genotyping perfor-
mance was achieved with greater than 90% call rate and 99.5% callaccuracy on over 90% of tested assays. The rhAmpTM genotypingsolution offers advanced bioinformatics driving a high assay designrate and minimal primer dimer formation, a dual enzyme master
dx.doi.org/10.1016/j.bdq.2017.02.047



dx.doi.org/10.1016/j.bdq.2017.02.048



dx.doi.org/10.1016/j.bdq.2017.02.049



dx.doi.org/10.1016/j.bdq.2017.02.050



S lar Det

msip

h

Sb

T

L

S

G

M

1

2

E

aopbpddrllmmhOVftceddaf

qs6

h


ix optimized for selectivity and stability, and an efficient univer-al reporter system that generates high fluorescent signal, offeringncreased confidence in SNP calling and the benefit of a lower coster genotype.


ession LTS-Genex – Technical Lunch-time Seminary MultiD & TATAA

ime: Tuesday, 04/Apr/2017: 1:00 pm–2:00 pm

ocation: Lecture hall 15, 350 participants – TUM Weihenstephan

ession Chair: Mikael Kubista, TATAA Biocenter AB, Sweden

enEx – the ultimate tool for qPCR data analysis

ikael Kubista 1,∗, Amin Forootan 2

TATAA Biocenter AB, Göteborg, SwedenMultiD Analyses AB, Göteborg, Sweden-mail address: [email protected]

GenEx was the first commercial software developed for qPCR,nd has since then become market leader with few thousandsf users and several thousands of publications using GenEx. Sup-orted by all leading qPCR instrument manufacturers GenEx hasatch import and corrects for inter-run variation. During pre-rocessing missing values are imputed, outliers are handled, andata are optimally normalized. Absolute quantification includesetermining limit of detection, limit of quantification and dynamicange. All parameters are determined with confidence ranges inine with CLSI, CEN and ISO guidelines. Expression data are ana-yzed with univariate methods including t-test, non-parametric

ethods and various ANOVAs with appropriate corrections forultiple testing ambiguity or multivariate methods including

ierarchical clustering, Principal Component Analysis (PCA), Self-rganizing Map (SOM), Artificial Neural Network (ANN), Supportector Machines (SVM), and many more. All analyses are easily per-

ormed with few clicks with the mouse using intuitive workflowshat generate results that are readily interpreted in a biologi-al context. At this lunch seminar, we will present GenEx andxemplify how easily multiple run files are in imported, measuredata pre-processed, and how data are interactively analyzed usingynamic PCA to reveal key expression pathways and correlationscross samples. All participants will receive FREE GenEx licenseor limited time. www.multid.se

The MIQE guidelines: minimum information for publication ofuantitative real-time PCR experiments. SA Bustin, V Benes, JA Gar-
on, J Hellemans, J Huggett, M Kubista, .Clinical chemistry 55 (4),11-622


Session NGS – Next Generation Sequencing



Session Chair: Justin O’Grady, University of East Anglia, UnitedKingdom

Session Chair: Dominik Buschmann, Technical University Munich,Germany

Improving the diagnosis and management ofserious infection using nanopore metagenomicsequencing

Justin O’Grady

University of East Anglia, United KingdomE-mail address: [email protected]

Rapid and accurate diagnosis is critical for the effective treat-ment of life threatening infections, such as bloodstream, respiratorytract and complicated urinary tract infections. These clinical syn-dromes have complex aetiology and require the recognition ofpathogens within challenging sample matrices. The “gold standard”culture techniques are labour intensive, have long turn-aroundtimes (≥2 days) and often offer poor clinical sensitivity. In theabsence of rapidly available pathogen or resistance information,the patient is treated empirically with broad spectrum therapy.This empirical therapy is often unnecessarily potent or, conversely,sometimes ineffective due to inherent or acquired resistance.

A paradigm shift in diagnostics technology is required, to allowthe development of a universal diagnostic which can detect anypathogen or resistance. Shotgun metagenomics sequencing has thepotential to drive this shift by combining rapidity with compre-hensiveness beyond that of culture or PCR. Nanopore real-timesequencing technology has, for the first time, made it feasible toapply metagenomic sequencing to acute infection diagnosis.

By providing rapid pathogen identification and AMR profiling,metagenomic sequencing will afford clinicians with a timely andcomprehensive diagnosis, reducing empiric treatment to a singledose and enabling tailored antimicrobial therapy. I will discuss ourongoing research on the development of metagenomic sequencingbased diagnostic tests for bloodstream, urinary tract and respira-tory tract infections.


Applications of an in vitro experimental modelfor a systems level understanding of health anddisease states of the human oral microbiome

Anna Elisabet Edlund 1,∗, Shibu Yooseph 2,Wenyuan Shi 3, Xuesong He 3,Jeffrey Scott McLean 4

1 J Craig Venter Institute, Genome Medicine Group,CA, USA2 University of Central Florida, College of Engineeringand Computer Science, FL, USA3 University of California Los Angeles, Shool of
Dentistry, CA, USA4 University of Washington, School of Dentistry, WA,USAE-mail address: [email protected]
dx.doi.org/10.1016/j.bdq.2017.02.051



http://www.multid.se/



dx.doi.org/10.1016/j.bdq.2017.02.052



dx.doi.org/10.1016/j.bdq.2017.02.053



lar Det

pamco(tiiotkocppmTt

h

Ac

BDLAS

1

2

3

E

aafd

dpttpptc

stmhtpwa

ev


Although oral microbial communities are subjected to dailyhysical and chemical disturbances such as fluctuations in pH,ntimicrobial agents, dietary components and personal hygieneeasures, a long-term stable microbiome persists. Biological pro-

esses that support this stability are important for the preventionf dysbiosis—a microbial shift toward a disease, e.g. periodontitisgum disease) or caries (tooth decay), the two most common infec-ious diseases of man. In our previous work, by studying an oraln vitro biofilm model system greater than 100 bacterial species, wedentified a plethora of metabolic activities possibly associated withral health both at the gene and molecule level. We also showedhat metabolic activities varied greatly for individual bacterialey-community members during pH fluctuations. Here, we focusn understanding molecular mechanisms and species interactionsritical for biofilm community stability during a 24-hour incubationeriod. By applying meta-omics approaches we dissect regulatoryathways that control the plankton-to-biofilm transition and theaintenance of the stable oral biofilm community during pH stress.

hese approaches also allow us to target gene transcription activi-ies of virulence mechanisms in low pH over time.


novel approach for selective enrichment ofustom gene targets for oncology research

joern Textor 1,∗, Andrew Barry 2,aniel Kraushaar 3, Sarah Bowman 3,ynne Apone 2, Kruti Patel 3, Noa Henig 3,my Emerman 3, Theodore Davis 2,alvaltore Russello 2, Cynthia Hendrickson 3

New England Biolabs GmbH, GermanyNew England Biolabs Inc., USADirected Genomics Inc., USA-mail address: [email protected]

Target enrichment of selected exonic regions for deep sequencenalysis is a widely used practice for the discovery of novel vari-nts, identification and phenotypic association of known variantsor a wide range of practical applications, including somatic variantetection in human cancer.

Deep sequencing of tumor material is required to effectivelyetect mutations that may be present at low frequencies, or in sam-les that contain a mixture of malignant and stromal cells. Due tohese challenges, highly focused gene panels are used to narrowhe regions being studied, reducing overall sequencing costs whileroviding the necessary coverage for variant detection. Challengesertaining to selecting the appropriate genes for inclusion are fur-her confounded by the high costs and time required to developustom gene panels.

The NEBNext DirectTM technology utilizes a novel approach toelectively enrich nucleic acid targets ranging from a single geneo several hundred genes, without sacrificing specificity. Further-

ore, intrinsic properties of the approach improve sensitivity andave proven amenable to challenging sample types including FFPEissue and circulating tumor DNA (ctDNA). The result is a 1-dayrotocol that enables the preparation of sequence-ready librariesith high specificity, uniformity, and sensitivity for the discovery

nd identification of nucleic acid variants.Here, we will discuss the NEBNext Direct approach to target

nrichment, specifically with regard to identification of somaticariants in clinically relevant samples, as well as content strategies


to efficiently customize content for genes involved in cancerresearch.


Liquid biopsies, biomarker signatures, andbeyond - why standardization of small RNA-Seqmatters

Dominik Buschmann 1,2,∗, Anna Haberberger 1,Benedikt Kirchner 1, Melanie Spornraft 1,Irmgard Riedmaier 4,5, Gustav Schelling 3,Michael W. Pfaffl 1

1 Department of Animal Physiology and Immunology,TUM School of Life Sciences Weihenstephan,Technical University of Munich, Freising, Germany2 Institute of Human Genetics, University Hospital,Ludwig-Maximilians-University, Munich, Germany3 Department of Anesthesiology, University Hospital,Ludwig-Maximilians-University, Munich, Germany4 Department of Physiology, TUM School of LifeSciences Weihenstephan, Technical University ofMunich, Freising, Germany5 Eurofins Medigenomix Forensik GmbH, Ebersberg,GermanyE-mail address: [email protected]

Small RNA-Seq has revolutionized transcriptomics research inmuch the same way RT-qPCR did several decades ago. The mas-sively parallel sequencing of short RNA reads has already yieldedunprecedented insights in areas such as gene expression pro-filing, clinical diagnostics, and biomarker discovery. Researchersare simultaneously intrigued by the technology’s promises, andchallenged with a multitude of hurdles on the way to accurateand meaningful data derived from high-throughput sequencing.Factors impacting experimental outcomes range from wet labparameters such as sample type and quality, RNA extraction, andlibrary preparation chemistry to processing and analysing mas-sive amounts of data in the post-experimental phase. In additionto the inherent complexity brought about by multiple types ofsmall RNA being studied in various biological scenarios, the choicefrom a plethora of commercially available sample processing kits,absence of comprehensive guidelines, and suboptimal reportingfurther complicate the body of literature. Reliable and reproducibleresearch findings can only be realized by rigorous experimentalstandardization and validation complemented with extensive andtransparent reporting of procedures in small RNA-Seq manuscripts.Forming hypotheses based on assumptions developed from flaweddata leads to inconsistent findings across the literature and impedestranslation of scientific discoveries into much-anticipated clini-cal applications such as molecular diagnostics and developmentof liquid biopsy-based disease biomarkers. Even though the paral-lels between RT-qPCR and RNA-Seq for nucleic acid quantificationare obvious, it could be argued that Next-Generation Sequencingtechniques bear even more risk for biases due to the complexityof post-experimental data processing. The widely-adopted MIQEguidelines clearly demonstrate how authoritative guidelines andquality standards translate into improved reporting, better exper-
imental setups and, ultimately, valuable applications of researchfindings. Focusing on the development of biomarker signatures,we herein similarly point out challenges along the small RNA-Seqworkflow, report common sources of experimental bias, and call
dx.doi.org/10.1016/j.bdq.2017.02.054



dx.doi.org/10.1016/j.bdq.2017.02.055



S lar Det

fh

h

Sq

YS

1

2

E

rttTtfctraplutlctpbte

h

F

LI

1

2

E

mcpsiuimoiTs

h

or biomarkers for disease diagnosis and treatment response moni-toring.



or rigorous quality control and validation in order to generateigh-quality, reproducible and meaningful sequencing data.


ingle-tube library prep solutions for highuality DNA sequencing

i Jin 1,2,∗, Marissa Bolduc 2, David Bays 2,huhong Li 2, Hongbo Liu 2, David Schuster 1,2

Quantabio, USAQIAGEN Beverly, USA-mail address: [email protected]

With wide adoption of NGS in research and healthcare, simple,apid, and reliable solutions for high quality NGS library prepara-ion are in great demand as library preparation can greatly impacthe sequencing results and overall sample to sequencing cost.o offer high-quality and streamlined library preparation solu-ions for the Illumina

®platform, we have developed two solutions

or different sequencing library preparations. The first solutionombines DNA fragmentation and library preparation in a singleube. This provides easily tunable enzymatic fragmentation, end-epair and dA-tailing in a single step, followed by high efficiencydapter ligation in the same reaction tube without interveningurification steps. The novel chemistry and simplified workflow

ead to DNA libraries with high complexity and even GC coverageniformity. For sample types which do not require DNA fragmen-ation, our second library prep product provides a fast, single tubeibrary preparation solution ensuring enhanced sensitivity and effi-iency, while maximizing library yields with minimal hands-onime. Leveraging our innovative chemistries, world-class enzymeurity and rigorously controlled production and quality system,oth single tube solutions offer unmatchable library prep resultshat address quality, speed, and throughput while remaining a cost-ffective option.


orensic application of microbiome profiling

isa-Marie Link 1,2, Jens Söchtig 2,rmgard Riedmaier-Sprenzel 2, Burkhard Rolf 2,∗

Eurofins Medigenomix GmbH, GermanyEurofins Medigenomix Forensik GmbH, Germany-mail address: [email protected]

The microbiome is defined as the collective genomes of theicrobes that live inside and on the human body. In a forensic

ontext, this is the collective genomes of the microbes that areresent in a stain sample. Investigating such samples with humanpecific STR-markers ignores the information that might be presentn this part of the extracted DNA. A forensic application could eitherse this for (additional) identification of the stain donor or for the

nvestigation of the source level of a stain. We investigated theicrobiomes of skin, mouth, nose and vaginal swabs from a set

f volunteers by V4 16S ribosomal RNA massive parallel sequenc-ng. Results of this study show the great diversity of the microflora.
he species observed by us in the various sample types overlap toome extent. Potential applications of the data are discussed.


Challenges of targeted NGS analyses

David Langenberger

ecSeq GmbH, GermanyE-mail address: [email protected]

Breast cancer is one of the most distributed and investigatedcancer types available and germline mutations in the BRCA1/2genes are known to lead to a high lifetime probability of devel-oping breast cancer. The application of targeted next-generationsequencing (NGS) to this cancer type has enabled the develop-ment of cost-effective and rapid tools for diagnostics and treatmentresponse/resistance. Even though available bioinformatics analysistools for detecting mutations in BRCA1/2 are highly developed andpopular, there are still issues with incorrectly called variants. Thistalk will cover some of these problems and suggest solutions fortargeted NGS data analysis.


Session ncRNA – non-coding RNAs


Location: Lecture hall 15, 350 participants –TUM Weihenstephan

Session Chair: Pieter Mestdagh, Ghent University, Belgium

Session Chair: Michael W Pfaffl, TUM, Germany

Cancer-specific long non-coding RNAs as novelbiomarkers and targets for therapy

Pieter Mestdagh

Ghent University, BelgiumE-mail address: [email protected]

Until recently, it was believed that only a small fraction of thegenome contained relevant information, used by the cell to pro-duce proteins. The majority was referred to as ‘junk DNA’ withno obvious function throughout life. The introduction of mas-sively parallel RNA-sequencing technology has drastically changedthat view. Today, there’s ample evidence demonstrating that themajority of the genome is transcribed, producing non-coding RNA(ncRNA) transcripts that differ in size, shape, expression and func-tion. The bulk of the non-coding transcriptome consists of so-calledlong non-coding RNAs (lncRNAs). These lncRNAs are character-ized by an exquisite tissue-specificity of lncRNAs which makesthem extremely attractive as targets for therapeutic intervention

dx.doi.org/10.1016/j.bdq.2017.02.056



dx.doi.org/10.1016/j.bdq.2017.02.057



dx.doi.org/10.1016/j.bdq.2017.02.058



dx.doi.org/10.1016/j.bdq.2017.02.059



dx.doi.org/10.1016/j.bdq.2017.02.060

lar Det

Tb

BD

LME

ssv‘c

mtboTgacsctgeetR

iudvi

h

ER

S

E

eshfmoo

psismgtl


he potentials of isomiRs – a new dimension iniomarker research

enedikt Kirchner, Ming Wu,ominik Buschmann, Michael W. Pfaffl ∗

Animal Physiology & Immunology, TUM School ofife Sciences Weihenstephan, Technical University ofunich, Freising, Germany

-mail address: [email protected]

Recent studies have proven that microvesicles are a valuableource of biomarkers in a number of physiological and pathophy-iological states. The molecular content of exosomes (and otheresicles) and their miRNA cargo in particular can act as a unique

biomarker signature’ and help identifying releasing cells and theirircumstances.

While knowledge of the extensive target repertoire of individualiRNAs greatly expanded our understanding of miRNA functions,

he high resolution down to single nucleotide alterations enabledy small-RNA sequencing technologies added an entire new layerf complexity by detecting a great abundance of miRNA isoforms.hese isomiRs show significant sequence and length heterogeneity,enerated by base exchanges from the canonical sequence, and/ordditions and/or deletions at the 3′ or 5′ end. The quantified isomiRsan change the mRNA targeting behaviour, either subtly throughupplementary or complementary binding at the 3′ end, or drasti-ally by altering the seed sequence at the 5′ end. By taking thesearget gene shifts into account and using a consensus of predictedenes that reflects actual miRNA sequence distribution and bindingfficiencies, the accuracy of predicted pathways and related dis-ases is greatly improved. To realize this approach we implementhese ideas in a ‘bioinformatical pipeline’ which analyses the smallNA sequencing read data files in a fully automatic way.

The assessment of mRNA targets can help discern the phys-ological relevance of identified miRNA biomarkers and evenncover novel relationships. We apply this ‘analysis pipeline’ toiscover new integrative mRNA/miRNA regulation pattern in agri-eterinary research and to predict new ‘miR biomarker signatures’n human clinical diagnostics.


pitranscriptomic regulation of non-codingNAs

tefan Ludwig Ameres

IMBA, Austria-mail address: [email protected]

For RNA to fulfil its essential function within the cellularnvironment, numerous chemical modifications have evolved toculpt its physical and functional interactions. Although more thanundred types of RNA modifications have built the descriptive

oundation of what is referred to as the epitranscriptome, theirode of action remains largely unknown. I will present our results

n the function of chemical RNA modifications at the intersectionf small RNA silencing pathways and general RNA metabolism.

Uridylation of RNA species represents an emerging theme inost-transcriptional gene regulation. In the microRNA pathway,uch modifications regulate small RNA biogenesis and stabilityn plants, worms, and mammals. We identified the first RNA-pecific uridylytransferase that is required for the majority of 3′ end
odifications of microRNAs in Drosophila and predominantly tar-
ets precursor hairpins. Uridylation modulates the characteristicwo-nucleotide 3′ overhang of microRNA hairpins, which regu-ates processing by Dicer and destabilizes RNA hairpins. Tailor


preferentially uridylates mirtron hairpins, thereby impeding theproduction of non-canonical microRNAs. Mirtron selectivity isexplained by primary sequence specificity of Tailor, selectingsubstrates ending with a 3′ guanosine. In contrast to mirtrons, con-served Drosophila precursor microRNAs are significantly depletedin 3′ guanosine, thereby escaping regulatory uridylation. Ourdata support the hypothesis that evolutionary adaptation toTailor-directed uridylation shapes the nucleotide composition ofprecursor microRNA 3′ ends. Hence, hairpin uridylation may serveas a barrier for the de novo creation of microRNAs in Drosophila.Our data also provide an atlas of post-transcriptional modificationsin small RNAs and their precursors in flies, providing a frameworkfor understanding the epitranscriptomic regulation of small RNAbiogenesis and function.

We could also show that uridylation in flies triggers the pro-cessive 3′-to-5′ exoribonucleolytic decay via the ribonucleaseII/R enzyme CG16940, a homolog of the human Perlman syn-drome exoribonuclease Dis3l2. Together with the TUTase Tailor,dmDis3l2 forms a stable cytoplasmic uridylation-triggered RNAprocessing (TRUMP) complex, that functionally cooperates in thedegradation of structured RNAs in vitro, providing a molecularexplanation for the inhibition of mirtron maturation in flies. RNA-immunoprecipitation and high-throughput sequencing reveals avariety of TRUMP complex substrates, including long non-codingRNA, such as rRNA, the essential RNase MRP and the signal recogni-tion particle RNA 7SL. Together with high-throughput biochemicalcharacterization of dmDis3l2 and bacterial RNase R our resultsimply a conserved molecular function of RNase II/R enzymes as‘readers’ of destabilizing post-transcriptional marks–uridylation ineukaryotes and adenylation in prokaryotes–that play importantroles in non-coding RNA surveillance.


Extracellular vesicles and their associatedcargos in health and disease

Mirco Castoldi

University of Düsseldorf, GermanyE-mail address: [email protected]

One of the major challenges towards an improved treatment ofhuman diseases is the identification of appropriate prognostic anddiagnostic markers. During the past decade, microRNA (miRNA)activity has been associated with the control of a wide range of cel-lular processes. Importantly, it has been shown that dysfunctionalexpression of specific miRNAs is associated with the developmentof a variety of diseases in human. However, the inherent difficul-ties associated to the collection of diseased material from patientshas severely limited the investigation of cellular miRNAs as sourcefor disease-related biomarker. Remarkably, the discovery of popu-lations of cell-free miRNAs circulating in the blood of healthy aswell as diseased individuals, have raised the possibility to iden-tify specific signature reflecting clinical manifestation of diseases.Although two different sub-populations of circulating miRNAsexist, we will specifically focus on the clinical utility, isolation,and detection of extracellular vesicles-associated miRNAs. Extra-cellular vesicles (EVs) are a heterogeneous group of nano-sizedparticles, which play a key role in inter-cellular communication andsignaling. EVs are secreted by most, if not all, cell types composingthe organism. Compelling evidences indicate that both the quan-titative and qualitative composition of EVs may correlate with the
diseased state of the patient. However, application of experimentalprotocols to the clinical setting is a challenge, requiring the estab-lishment of easy to use, affordable and robust methodologies. Here,


dx.doi.org/10.1016/j.bdq.2017.02.061



dx.doi.org/10.1016/j.bdq.2017.02.062



S lar Det

itsa

h

ITb

A

E

dsipd

(t

(so(st

h

Aam

S

E

vbmstmtabAhtwcbSrps

h


mplementation of experimental pipelines required for the isola-ion and analysis of EVs and EVs-associated miRNAs from patients’erum in the context of diagnostic laboratories will be presentednd discussed.


n silico functional analysis of microRNA:owards the characterisation of miRNAs asiomarkers

rtemis Hatzigeorgiou

University of Thessaly, Greece-mail address: [email protected]

RNA revolution has turned non-coding RNAs (ncRNAs) fromark-matter into a biological research hotspot. ncRNA families,uch as microRNAs (miRNAs) and more recently, long non cod-ng RNAs (lncRNAs) are being researched for physiological andathological implications. The role of miRNAs in development andiseasehas been widely reported during the past few years.

During the talk tools from the DIANA suite of miRNA analysismicrorna.gr) will be presented that can be used as basic step forhe identification of miRNA biomarkers.

Such tools and databases include miRNA target predictionDIANA micro-T) and annotation (TarBase, LNCBase), pathway analy-is (DIANA–mirPath), regulation of microRNAs (miRGEN) and the usef RNAseq data for the identification of key regulatory componentsmiRNA and/or Transcription factors) based on two investigatedtages (DIANA- mirExTra). The DIANA suite is widely used with morehan 100,000 unique users per year from all over the world.


novel assay system for improved specificitynd sensitivity of miRNA detection for all theajor model organisms

imon Baker

Bioline Reagents Limited, United Kingdom-mail address: [email protected]

For some time now, Bioline has offered an extensive list of indi-idual human miRNAs, using the proprietary algorithm developedy MiRXES to maximize miRNA detection sensitivity, while mini-izing non-specific interactions. The versatility of the assay design

ystem has now allowed us to extend the range to include allhe 27,000 miRNA listed on miRBase, providing plant and animal

iRNA assays. The resulting real-time PCR assays enable detec-ion of extremely low levels of miRNA with high specificity usingSYBR® Green detection chemistry, allowing the discrimination

etween closely related miRNA sequences. All EPIK miRNA Selectssays have been validated using synthetic miRNA templates anduman assays have also been validated against total RNA. Typicallyhe assays detect as few as 100 copies of template per RT reactionith excellent assay efficiency and linearity. We will also dis-

uss the appropriate uses of positive controls to normalize resultsetween assays and between experiments, and how the EPIK RNApike-In controls can be used to build an accurate picture of theelative concentration of miRNA in a sample. These controls can bearticularly effective in addressing complex biological problemsuch as the abundance of miRNA in exosomes from human blood.



Two-tailed RT-qPCR: A novel method for highlyaccurate MiRNA quantification

Lukas Valihrach 1,∗, Peter Androvic 1, Julie Elling 2,Robert Sjoback 2, Mikael Kubista 1,2

1 Institute of Biotechnology AS CR, Czech Republic2 TATAA Biocenter AB, SwedenE-mail address: [email protected]

MicroRNAs are a class of small non-coding RNAs that serve asimportant regulators of gene expression at the posttranscriptionallevel. MiRNAs are stable in body fluids and pose great potentialto serve as biomarkers. Here, we present a highly specific, sensi-tive and cost-effective system to quantify miRNA expression basedon two-step RT-qPCR with SYBR-green detection chemistry calledTwo-tailed RT-qPCR. It takes advantage of novel, target-specificprimers for reverse transcription composed of two hemiprobescomplementary to two different parts of the targeted miRNA,connected by a hairpin structure. The introduction of a secondprobe ensures high sensitivity and enables discrimination of highlyhomologous miRNAs irrespectively of the position of the mis-matched nucleotide. Two-tailed RT-qPCR has a dynamic rangeof 8 logs and a sensitivity sufficient to detect down to a hun-dred of target miRNA molecules. It is capable to capture the fullisomiR repertoire, leading to accurate representation of the com-plete miRNA content in a sample. The reverse transcription step canbe multiplexed and the miRNA profiles measured with Two-tailedRT-qPCR show excellent correlation with the industry standardTaqMan miRNA assays (R2 = 0.985). Moreover, Two-tailed RT-qPCRallows for rapid testing with a total analysis time of less than2.5 hours.


Session MIQE&QC – MIQE & qPCR Quality Control

Time: Wednesday, 05/Apr/2017: 9:00 am–12:30 pm


Session Chair: Stephen Andrew Bustin, Anglia Ruskin University,United Kingdom

Session Chair: Jan M Ruijter, Academic Medical Center, TheNetherlands

Why are we still using qPCR for thequantification of nucleic acids?

Stephen Andrew Bustin

Anglia Ruskin University, United KingdomE-mail address: [email protected]

Real-time fluorescence-dependent quantitative PCR, with orwithout a preceding reverse transcription (RT) step, has long beenembraced as a valuable and valid means for quantifying nucleicacids. Its results are widely used to report molecular biomarkers ofdisease, quantify changes to RNA levels in cells or biopsies and iden-tify the tissue of origin from deposits on forensic samples. Clearly,qPCR has an important and effective use for detecting pathogens,
SNPs or other DNA-associated applications. However, this accep-tance for RNA is remarkable, given the numerous reports that haveemerged over the last twenty years or so that should cast significantdoubts on how reliable RT-qPCR data really are. Whilst RT-qPCR
dx.doi.org/10.1016/j.bdq.2017.02.063



dx.doi.org/10.1016/j.bdq.2017.02.064



dx.doi.org/10.1016/j.bdq.2017.02.065



dx.doi.org/10.1016/j.bdq.2017.02.066



lar Det

ccosti

h

Tr

JAS

1

M2

BC3

U4

AE

boTaqtuopgoacad(it

N

w

N

i

N

t

0

n(vwvw


an undoubtedly be used to distinguish fairly large differences orhanges in nucleic acid levels, the majority of conclusions are basedn results that report small changes that are neither robust nor con-istent. This raises the far-reaching question of whether it is timeo abandon RT-qPCR as a method for quantifying RNAs and replacet with more accurate and less error-prone digital PCR technology.


arget concentration and replicate qPCReactions

an M. Ruijter 1,∗, Maurice W.J. de Ronde 2,ntoni Bayes Genis 3, Yigal Pinto 4,ara-Joan Pinto 2

Anatomy, Embryology and Physiology, Academicedical Center, Amsterdam, the NetherlandsVascular Medicine & Clinical Epidemiology,iostatistics and Bioinformatics, Academic Medicalenter, Amsterdam, the NetherlandsHeart Failure Unit, Germans Trias i Pujol Hospital,niversitat Autònoma, Barcelona, SpainExperimental Cardiology, Academic Medical Center,msterdam, the Netherlands-mail address: [email protected]

In the analysis of qPCR data, the criterion for reproducibilityetween replicate reactions is set to 0.5 cycles; when the Cq valuesf replicates differ more than 0.5, the results are often discarded.he idea is that this rule-of-thumb should be enforced to protectgainst the inclusion of technical variation in the analysis of thePCR results. However, a technical pipetting error of 15% is requiredo reach a 0.5 cycles difference. Smaller technical errors are alreadynacceptably large but would go unnoticed and be tolerated. On thether hand, at high Cq values the sampling error that occurs whenipetting a low number of target molecules into the PCR plate isoverned by the Poisson distribution. To calculate the magnitudef this sampling error for different Cq values, we started with thessumption that for a PCR efficiency of about 1.8, an input of 10opies of target leads to a Cq value of about 35 when the primer andmplicon concentration become similar. The observed Cq value willepend on the actual PCR efficiency, the monitoring chemistry, thepre-) processing of the fluorescence data and the way the Cq values determined. When the PCR reaction reaches the quantificationhreshold the general kinetic equation can be written as

q = NECq (1)

hich with the above assumption is equal to

q = 10E35 (2)

This equality provides us with an equation to estimate the targetnput from the observed Cq value and PCR efficiency:

q = 10E35-Cq (3)

Using the relationship between Poisson and Chi2 distributionshe 95% confidence interval of N is then given by:

.5�20.025;2N ≤ N ≤ 0.5�2

0.975;2N+2 (4)

For each Cq and PCR efficiency value we can thus calculate aumber of copies with Eq. (3), the sampling error range with Eq.4) and then, with the inverse of Eq. (1), the expected range of Cq

alues due to unavoidable sampling variation. This range increasesith higher Cq values (less target) and easily leads to a replicate

ariation above 0.5 cycles. Discarding such discordant replicatesill lead to bias and loss of power in the analysis. For a dataset that


resulted from the qPCR measurement of 12 miRNA targets in 834patients with a total of 20,016 reactions, the strict application of therule that the Cq values of replicates should be within 0.5 cycles led torejection of 7752 (39%) measurements. When the unavoidable Cq

range, calculated as described above, is used to determine whichCq difference between replicates should lead to the decision thatthe replicate discordance is still acceptable, the number of reac-tions that should be discarded decreases to 1414 (7%). Not only lessreactions are discarded, also the distribution of Cq values changesshowing that strict application of the Cq < 0.5 cycle rule has led tobias in the results of the qPCR experiment. A decision to excludereplicate reactions based on the expected sampling error avoidsthis bias, prevents unwanted loss of data and therefore increasesthe statistical power.


Laboratory automation and data managementin diagnostics – qPCR ZIKV detection andquantification

Klemen Zupancic 1, Misa Korva 2,Tatjana Avsic Zupanc 2, Urska Cepin 3,Manca Pirc 3, Laura Simdon 4, Matjaz Hren 1,3,∗

1 SCINOTE, LLC, USA2 Institute of Microbiology and Immunology, Facultyof Medicine, University of Ljubljana, Slovenia3 BioSistemika, d.o.o., Slovenia4 Gilson, Inc., USAE-mail address: [email protected]

Automation has significantly improved human diagnosticsin the developed world by increasing patient throughput anddecreasing diagnostic variability caused by human interaction. Theoutbreak and expansion of Zika virus (ZIKV) has increased the needfor rapid and reliable diagnostics in the early stages of infection andfor virus quantification in clinical studies. Real time PCR (qPCR) hasbeen found to be the most sensitive, specific and rapid detectionsystem for ZIKV detection.

Our study addressed automation of qPCR plate setup for ZIKVdetection and quantification which includes management of sam-ples, preparation of sample dilutions, preparation of master mixand their application to the qPCR plate. This was compared tomanual qPCR setup which was performed by an experienced labdiagnostician.

In this study samples were tested from five human patients withsuspicion on ZIKV infection that were sent for routine diagnosticsof ZIKV to the Institute of Microbiology and Immunology, Facultyof Medicine, University of Ljubljana, Slovenia (IMI). RNA was iso-lated from different tissues or fluids including fetus brain, blood,semen, plasma, or urine. Additionally, for an absolute quantifica-tion study, six different ZIKV strains maintained in cell culture lineswere analysed. Necessary controls were included in every step ofthe process. The same samples were used for manual and auto-mated qPCR experiments on the same day.

The automated setup included management of samples insciNote Open Source Electronic Lab Notebook while sample dilu-tions, master mix preparation and qPCR plate setup was done byusing Gilson qPCR Assistant with PIPETMAX®268 automated pipet-ting workstation (PIPETMAX®).

Analysis of the qPCR data revealed that the accuracy of perfor-mance of automated qPCR plate setup was comparable to manual
setup done by a very experienced lab diagnostician while the speedof the setup was improved by automating the sample import, sam-ple dilutions and qPCR plate setup.
dx.doi.org/10.1016/j.bdq.2017.02.067



dx.doi.org/10.1016/j.bdq.2017.02.068



S lar Det

tsMatsr

h

Vld

MJL

1

2

3

E

mo3wuHtcrr

q

C

h

Iop

G

E

ottWsq

p1t

de

for interplate variation to perform high quality quantitative PCRmeasurements.



Incorporating an automation platform into the diagnostic pro-ocol allows for accurate standard dilution and assay setup with aignificant increase in throughput compared to manual processing.oreover, integration of data management software with an

utomation system further increases the throughput and reduceshe possibility of user error. More importantly, data managementoftware, such as sciNote, enables full traceability of samples andesults which is critical for accuracy in human diagnostics.


alidation of a qPCR method – Determiningimit of detection, limit of quantification andynamic range

ikael Kubista 1,∗, Robert Sjöback 1,ens Björkman 1, Björn Sjögreen 2,ucas Luke Linz 3, Amin Forootan 2

TATAA Biocenter AB, SwedenMultiD Analyses AB, SwedenLGC Douglas Scientific, USA-mail address: [email protected]

Quantitative Real-Time Polymerase Chain Reaction (qPCR) is theost sensitive and specific technique we have for the detection

f nucleic acids. Even though it has been around for more than0 years and is preferred in research applications, it has yet toin broad acceptance in routine practice. This requires a means tonambiguously assess the performance of specific qPCR analyses.ere we present methods to determine the limit of detection (LoD),

he limit of quantification (LoQ) and the Dynamic Range as appli-able to qPCR. These are based on standard statistical methods asecommended by regulatory bodies and adapted to the logarithmicesponse characteristic of qPCR.

The MIQE guidelines: minimum information for publication ofuantitative real-time PCR experiments

SA Bustin, V Benes, JA Garson, J Hellemans, J Huggett, M Kubista,linical chemistry 55 (4), 611-622

Prime time for qPCR–raising the quality barM Kubista Eur. Pharm. Rev. 19, 60-67


nfluence of PCR consumables on the accuracyf real-time PCR experiments and NGS samplereparation

errit Gutzke, Hanna Oldfield ∗, Emily Flowers

4titude Ltd, Germany-mail address: [email protected]

Classical PCR and qPCR plates are one-component plates madeut of polypropylene (PP). PP is the best plastic material for PCRubes as it is chemically inert and allows for the production of ultra-hin tube walls which is important for fast temperature transfer.

hile PP has become the standard material for PCR consumablesome of its properties question its suitability for applications likePCR and NGS:

The material characteristics of PP exhibit a Vicat Softening Tem-erature (VST) of 90 ◦C and a coefficient of thermal expansion of80 × 10−6 K−1 which are potential weaknesses for its usage atypical (q)PCR temperatures.

When used for (q)PCR, not only do the plates soften during theenaturation step, but measurements also show that the platesxpand by up to 2 mm in the diagonal plane (from room tempera-


ture to 95 ◦C) and they shrink again as the temperature decreases.Therefore, the plate will undergo expansion and contraction inevery cycle, placing significant tension on the plate seals. As a result,contact between the seal and plate will be particularly weakenedin the corner positions and outer rows leading to evaporation fromthe plate in these areas, while centre wells will only be affectedminimally. This differential evaporation effect is especially eminentwhen adhesive seals are used (as opposed to heat seals).

Evaporation has a significant effect on the reaction conditionsresulting in noticeable effects, especially for qPCR. Identical sam-ples can exhibit significant differences in their Ct values, dependingon their position on the plate. This often remains unnoticed as trip-licates are typically placed in neighbouring wells which are affectedby similar levels of evaporation.

A solution to the problem of evaporation related qPCR inaccu-racies is the usage of two-component plates. These plates consistof tubes made out of PP but a frame made out of polycarbonate(PC). PC does not show significant temperature-dependant expan-sion and contraction as the VST of this material is 147 ◦C and thecoefficient of thermal expansion is 70 × 10−6 K−1.

The ongoing trend to continually reduce DNA concentrationscan make additional modifications or choices necessary. DNA hasbeen shown to bind to PP tubes in trace amounts, especially inhighly ionic conditions. Different PP polymers are used for the pro-duction of PCR consumables as they differ in their characteristics,such as surface charge. As a result, different PCR plates bind DNA invarying amounts. Furthermore, the commercially used term “low-binding” is poorly defined.

DNA binding to PP surfaces has not been reported as an issuefor PCR/qPCR as DNA stuck to the walls will be released duringdenaturation steps. However, NGS protocols contain a number oftransfer steps from one tube to another and ultra-low DNA bindingcharacteristics may be required if only trace amounts of nucleicacids are used.


How to monitor analytical and technical factorsinfluencing qPCR

Jens Bjorkman

TATAA Biocenter, SwedenE-mail address: [email protected]

qPCR is today a mature and most established method. Still it iseasy to neglect the underlying factors that may compromise theresults. With an easy-to-use highly automated qPCR instrument inevery lab, scientist can (if they choose) generate data with mini-mum focus on the pre-qPCR parameters that may have significantimpact on the data. It is not difficult to produce Cq-values, but howdo we know that they truly reflect the amount of target that wasactually present in the original sample or even in vivo? As describedin the MIQE guidelines proper controls should be included in orderto identify and in some cases correct for the bias caused by pre-analytical parameters and technical variation. I will describe qualitycontrol measures to test for degradation of RNA, RT and PCR inhibi-tion, genomic DNA background, and provide means to compensate

dx.doi.org/10.1016/j.bdq.2017.02.069



dx.doi.org/10.1016/j.bdq.2017.02.070



dx.doi.org/10.1016/j.bdq.2017.02.071



dx.doi.org/10.1016/j.bdq.2017.02.072

lar Det

S

T

L

SL

S

DdMc

DICMBPLRM

1

HT2

S3

P4

T5

eE

msafcistMf

lwsa(((aow

afiula


ession dPCR 2 – digital PCR 2

ime: Wednesday, 05/Apr/2017: 9:00 am–12:30 pm


ession Chair: Afif M. Abdel Nour, Holy Spirit University of Kaslik,ebanon (Lebanese Republic)

ession Chair: David Svec, TATAA Biocenter, Czech Republic

roplet digital PCR for MYD88L265P mutationetection in Waldenström Macroglobulinemia:inimal residual disease monitoring and

haracterization on circulating free DNA

aniela Drandi 1,∗, Elisa Genuardi 1,rene Dogliotti 1, Martina Ferrante 1,ristina Jimenez 2, Francesca Guerrini 3,ariella Lo Schirico 1, Vittorio Muccio 1,

arbara Mantoan 1, Milena Gilestro 4,aola Omedè 4, Sara Galimberti 3,orella Orsucci 4, Federica Cavallo 1,amon Garcia Sanz 2, Mario Boccadoro 1,4,arco Ladetto 5, Simone Ferrero 1

Department of Molecular Biotechnologies andealth Sciences, Hematology Division, University oforino, ItalyServicio de Hematología, Hospital Universitario dealamanca, Salamanca, SpainClinical and Experimental Medicine, University ofisa, Pisa, ItalyDivision of Hematology, ASO S.Giovanni Battista,orino, ItalyDivision of Hematology, Az Ospedaliera SS AntonioBiagio e Cesare Arrigo, Alessandria, Italy-mail address: [email protected]

Background: MYD88L265P mutation might represent an idealarker for minimal residual disease (MRD) monitoring in Walden-

tröm Macroglobulinemia (WM). However, the conventionalllele-specific quantitative PCR (ASqPCR) is not sensitive enoughor MRD monitoring on peripheral blood (PB), harboring lowoncentrations of tumor cells. Besides, cell-free DNA (cfDNA) isncreasingly used for mutational studies. We set up a new, highlyensitive, droplet digital PCR (ddPCR) assay for MYD88L265P detec-ion and described: 1) its feasibility for mutation screening and

RD monitoring in bone marrow (BM) and PB; 2) its applicationor mutational studies on cfDNA.

Methods: BM, PB and plasma from local series of WM, IgG-ymphoplasmacytic lymphoma (LPL) and IgM-MGUS patients (pts)

ere collected at baseline and during follow-up (FU). 40 healthyubjects were used as negative controls. Genomic DNA (gDNA)nd plasmatic cfDNA were extracted by Maxwell RSC systemPromega). MYD88L265P was assessed on gDNA (100 ng) and cfDNAfrom 1 ml of plasma) by a custom ddPCR assay on a QX100 SystemBio-Rad). For comparison ASqPCR was assessed on gDNA (100 ng),s described [Xu L, 2013]. MYD88L265P cut-off was settled basedn the healthy samples background level. IGH-based MRD analysisas performed as described [Drandi D, 2015].

Results: Sensitivity of ddPCR versus ASqPCR was assessed onten-fold serial dilution standard curve. Whereas ASqPCR con-

rmed the sensitivity of 1.00E−03, ddPCR reached a sensitivity
p to 5.00E−05. Overall, 291 samples from 148 pts, 194 base-
ine (128 BM, 66 PB) and 97 follow-up (43 BM and 54 PB), werenalyzed. 123/128 (96.1%) diagnostic BM and 47/66 (71.2%) PB


samples scored positive for MYD88L265P (BM median 3.6%, range:0.02-72.6%: PB median 0.3%, range: 0.01-27.8%). 11/46 (24%) ptswith both BM/PB collected at diagnosis showed a positive/negativematch. Concordances between ddPCR and qPCR methods wereinvestigate on 100 samples (60 BM, 40 PB) and overall a goodconcordance was observed (p = 0.0005). Of note the majority ofdiscordances were observed in the follow-up samples. Moreover,to investigate whether MYD88L265P ddPCR could be used for MRDmonitoring we compared it to the gold standard IGH-based MRDassay in baseline and FU samples (23 BM, 15 PB) from 10 pts. Thecomparison showed highly superimposable results between meth-ods. Finally, ddPCR performed on cfDNA from 60 plasma samplesshowed 1 log higher median levels of MYD88L265P mutation bymutation in plasma (1.4%, range 0–72.2%) compared to PB (0.1%,range: 0.01–27.8%).

Conclusion: ddPCR is a feasible and highly sensitive assay formutational screening and MRD monitoring in WM, particularly insamples harboring low concentrations of circulating tumor cells.Moreover, plasmatic cfDNA represents a promising tissue sourceand might be an attractive, less invasive alternative to PB or BM forMYD88L265P detection.


Partition volume in dPCR – Monodispersity notreally important

Joel Tellinghuisen

Vanderbilt University, USAE-mail address: [email protected]

The role of partition volume variability, or polydispersity, indigital polymerase chain reaction methods is examined throughformal considerations and Monte Carlo simulations. Contrary tointuition, polydispersity causes little precision loss for low averagecopy number per partition � and can actually improve precisionwhen � exceeds ∼4. It does this by negatively biasing the esti-mates of �, thus increasing the number of negative (null) partitionsN0. In keeping with binomial statistics, this increases the relativeprecision of N0 and hence of the biased estimate m of �. Below� = 1, the precision loss and the bias are both small enough to benegligible for many applications. For higher � the bias becomesmore important than the imprecision, making accuracy depend-ent on knowledge of the partition volume distribution function.This information can be gained with optical microscopy or throughcalibration with reference materials.


Automated cell picking and single cell digitalPCR focusing on mitochondrial transfer

David Svec 1,2

1 Institute of Biotechnology AS CR, Prague2 TATAA Biocenter, Czech RepublicE-mail address: [email protected]

Mitochondria are unique organelles comprising their owngenetic information in the form of mitochondrial DNA (mtDNA).Cell type mtDNA heteroplasmy was reported before as a commonevent observed not only in patients with mitochondrial diseases butalso in healthy individuals. In parallel reports show that mitochon-
dria move between mammalian cells. We performed single cellexpression profiling focusing on horizontal mitochondrial transfer.Using automated cell picking and single cell digital PCR, we showed


dx.doi.org/10.1016/j.bdq.2017.02.073



dx.doi.org/10.1016/j.bdq.2017.02.074



S lar Det

tcm

h

Dsb

MRPJ

1

L2

L3

IM4

UGE

amncHstpgPdiaficswotcsuspdR

h


hat generation of tumors in syngeneic mice by cells devoid of mito-hondrial (mt) DNA (�0 cells) is linked to acquisition of the hosttDNA, leading to the normalization of mitochondrial respiration.


igital PCR inhibition mechanisms usingtandardized inhibitors representing soil andlood matrices

aja Sidstedt 1,2,∗, Erica L. Romsos 3,onny Hedell 2,4, Carolyn R. Steffen 3,eter M. Vallone 3, Peter Rådström 1,ohannes Hedman 1,2

Applied Microbiology, Department of Chemistry,und University, PO Box 124, 221 00 Lund, SwedenSwedish National Forensic Centre, 581 94inköping, SwedenMaterials Measurement Laboratory, National

nstitute of Standards and Technology, Gaithersburg,D 20899-8314, United StatesDepartment of Mathematical Sciences, Chalmersniversity of Technology and University ofothenburg, 41296 Gothenburg, Sweden-mail address: [email protected]

Digital PCR (dPCR) enables absolute quantification of nucleiccids by partitioning the sample into hundreds or thousands ofinute reactions [1]. By assuming a Poisson distribution for the

umber of DNA fragments present in each chamber, the DNA con-entration is determined without the need for a standard curve.owever, when analyzing nucleic acids from complex matrices

uch as soil and blood, the dPCR quantification can be biased dueo the presence of inhibitory compounds [2,3]. In this study, weresent how certain inhibitors disturb dPCR quantification and sug-est solutions to these problems. Furthermore, we use real-timeCR, dPCR and isothermal titration calorimetry as tools to eluci-ate the mechanisms underlying the PCR inhibition. The impact of

mpurities on dPCR quantification was studied using humic acids a model inhibitor. We show that the inhibitor-tolerance dif-ers greatly for three different DNA polymerases, illustrating themportance of choosing a DNA polymerase-buffer system that isompatible with the samples to be analysed. Various inhibitory-ubstances from blood were found to disturb the system in differentays. For example, hemoglobin was found to cause quenching

f fluorescence and a dramatic decrease of the number of posi-ive reactions, leading to an underestimation of DNA quantity. IgGaused an increased number of late-starters. The system was moreusceptible to inhibition by IgG when single-stranded DNA wassed as template, compared with double-stranded DNA. By under-tanding more about the mechanisms of PCR inhibitors it will beossible to design more optimal PCR chemistries, improving dPCRetection and quantification.eferences

1 M. Baker, Digital PCR hits its stride, Nat. Methods 9 (2012) 541–544.2 C. Coudray-Meunier, A. Fraisse, S. Martin-Latil, L. Guillier, S. Delannoy, P. Fach,

S. Perelle, A comparative study of digital RT-PCR and RT-qPCR forquantification of Hepatitis A virus and Norovirus in lettuce and water samples,Int. J. Food Microbiol. 201 (2015) 17–26.

3 T. Hoshino, F. Inagaki, Molecular quantification of environmental DNA usingmicrofluidics and digital PCR, Syst. Appl. Microbiol. 35 (2012) 390–395.



dpcReport: Web server and software suite forunified analysis of digital PCRs and digitalassays

Michał Burdukiewicz 1,∗, Jim Hugget 2,Alexandra Whale 2, Boris Fehse 3, Piotr Sobczyk 4,Paweł Mackiewicz 1, Andrej-Nikolai Spiess 5,Peter Schierack 6, Stefan Rödiger 6

1 Department of Genomics, University of Wrocław,Poland2 Molecular and Cell Biology Team, LGC, Teddington,United Kingdom3 Research Department Cell and Gene Therapy,Department of SCT, University Medical CenterHamburg-Eppendorf, Hamburg, Germany4 Faculty of Pure and Applied Mathematics, WrocławUniversity of Science and Technology, Wrocław,Poland5 University Medical Center Hamburg-Eppendorf,Hamburg, Germany6 Institute of Biotechnology, Brandenburg Universityof Technology Cottbus-Senftenberg, Senftenberg,GermanyE-mail address: [email protected]

Digital PCR (dPCR) is a variant of PCR, where the PCR ampli-fication is conducted in multiple small volume reactions (termedpartitions) instead of a bulk. The dichotomous status of each par-tition (positive or negative amplification) is used for absolutequantification of the template molecules by Poisson transforma-tion of the proportion of positive partitions. The vast expansionof dPCR technology and its applications has been followed by thedevelopment of statistical data analysis methods. Yet, the softwarelandscape is scattered, consisting of scripts in various programminglanguages, web servers with narrow scopes or closed source vendorsoftware packages, that are usually tightly tied to their platform.This leads to unfavourable environments, as results from differ-ent platforms, or even from different laboratories using the sameplatform, cannot be easily compared with one another.

To address these problems, we developed the dpcReport webserver that provides an open-source tool for the analysis of dPCRdata. dpcReport provides a streamlined analysis framework to thedPCR community that is compatible with the data output (e.g.,CSV, XLSX) from different dPCR platforms (e.g., Bio-Rad QX100/200,Biomark). This goes beyond the basic dPCR data analysis withvendor-supplied softwares, which is often limited to the compu-tation of the mean template copy number per partition and itsuncertainty. dpcReport gives users more control over their dataanalysis and they benefit from standardization and reproducibleanalysis.

Our web server analyses data regardless of the platform ven-dor or type (droplet or chamber dPCR). It is not limited to thecommercially available platforms and can also be used with exper-imental systems by importing data through the universal REDFformat, which follows the IETF® RFC 4180 standard. dpcReportprovides users with advanced tools for data quality control andit incorporates statistical tests for comparing multiple reactionsin an experiment, currently absent in many dPCR-related soft-ware tools. dpcReport provides users with advanced tools for dataquality control. The conducted analyses are fully integrated withinextensive and customizable interactive HTML reports includingfigures, tables and calculations. To improve reproducibility and
transparency, a report may include snippets in the programminglanguage R enabling an exact reproduction of the analysis per-formed by dpcReport through functions from the dpcR package.Our software follows the standardized dPCR nomenclature of the
dx.doi.org/10.1016/j.bdq.2017.02.075



dx.doi.org/10.1016/j.bdq.2017.02.076



lar Det

dpd

w

h

Ac

AM

E

qotctapTuartmpmu2rtoticTBtWtdlccdflrmnrTap

h


MIQE guidelines. Since the vast functionality offered by dpcRe-ort may be overwhelming at first, our web server is extensivelyocumented.

The server is freely accessible at: http://www.smorfland.uni.roc.pl/shiny/dpcReport/.


statistical contribution to the uncertainty ofoncentration measurements using digital PCR

ndreas Kummrow, Annabell Plauth ∗,artin Hussels

Physikalisch-Technische Bundesanstalt, Germany-mail address: [email protected]

Measurement of the concentration of biological entities usinguantitative PCR (qPCR) usually results in a large spread of resultsbtained in interlaboratory comparisons. As such, deviations inhe range of ±0.6 on a logarithmic scale (base 10) are currentlyonsidered acceptable in the external quality assurance of quan-ification of viruses like HBV, HCV, and HIV-1 in Germany. Notably,

considerable contribution to this variance may originate fromre-analytical steps and variable efficacy of the assays utilized.echnical limitations of the instrument remain as a source ofncertainty of measurement, even if a perfect extraction andmplification is assumed. Digital PCR (dPCR) does neither requireeference genes nor calibration material to quantify the concentra-ion in biological samples and thus might substantially improve the

easurement uncertainty in concentration measurements com-ared to qPCR. An apparent physical limitation for concentrationeasurements using dPCR is the uncertainty of the reaction vol-

me. Recent studies quote an (unexpanded) uncertainty of around% using microscopic measurements of the average size of theespective reaction volume. In this study, we discuss the effect ofhe limited number of repeat reactions in dPCR on the uncertaintyf concentration. It is generally accepted that a Poisson correc-ion must be applied to the concentration determined by dPCR,.e. for an average of � reactive DNA molecules in each reactionhamber, the probability of a positive reaction is p = (1 − exp(−�)).hus, for N reactions one expects A = pN positive reactions and= (1 − p)N negative reactions. The statistical uncertainty for posi-

ive (and negative) reactions does not follow a normal distribution.e show, that the uncertainty of finding positive reactions has

o be calculated based on the standard deviation of the binomialistribution, which yields u = (AB/N)1/2. This result is used to calcu-

ate the minimum number of reactions required for a given DNAoncentration � and targeted statistical uncertainty u of the con-entration. Additionally, the implications of this result on setting upPCR experiments is discussed. We calculated the sweet spot for theewest number of reactions around � = 1.5, which falls to � = 1 forarge statistical uncertainties (>10%). Even in this case, at least 4000eactions are required to reach a statistical uncertainty of 2%. Toaintain this uncertainty for a dynamic range of 3 orders of mag-

itude requires using more than 300000 reactions. The researcheceives funding from the EMPIR project HLT-07 AntiMicroResist.he EMPIR programme is co-financed by the Participating Statesnd the European Union’s Horizon 2020 research and innovationrogramme.



Session LTS-Kramer – Technical Lunch-time Semi-nar by Kramer

Time: Wednesday, 05/Apr/2017: 12:30 pm–1:30 pm

Location: Lecture hall 14, 650 participants - TUM Weihenstephan

Session Chair: Fred Russell Kramer, Rutgers University, UnitedStates of America

Multiplex real-time PCR assays that measurethe abundance of extremely rare mutationsassociated with cancer

Fred Russell Kramer

Rutgers University, USAE-mail address: [email protected]

PCR assays are the most rapid, most sensitive, and least expen-sive way to assess the abundance of mutant DNA fragments presentin liquid biopsies. “SuperSelective” PCR primers, due to their uniquedesign, are extraordinarily specific, able to selectively initiate thesynthesis of amplicons on ten mutant DNA fragments in thepresence of 1,000,000 wild-type DNA fragments. Sets of SuperSe-lective primers, each possessing unique 5′-tag sequences, enablethe amplicons generated from each mutant to be distinguishedby differently colored molecular beacon probes. And the inclusionof primers for a wild-type reference gene fragment, enables theabundance of each type of mutant DNA fragment to be assessedby determining the difference between its threshold value and thethreshold value of the reference gene.


Session DA – qPCR Data Analysis

Time: Wednesday, 05/Apr/2017: 2:00 pm–4:30 pm


Session Chair: Stefan Rödiger, BTU Cottbus - Senftenberg,Germany

Session Chair: Andrej-Nikolai Spiess, University HospitalHamburg-Eppendorf, Germany

Methods and technologies for analysis digitalPCR experiments – Lessons we learned from theqPCR technology

Stefan Rödiger 1,∗, Andrej-Nikolai Spiess 2,Michał Burdukiewicz 3

1 Brandenburg University of TechnologyCottbus–Senftenberg, Senftenberg, Germany2 University Medical Center Hamburg-Eppendorf,Hamburg, Germany3 University of Wroclaw, Wroclaw, PolandE-mail address: [email protected]

Quantitative PCR (qPCR) and digital PCR (dPCR) are widely
employed DNA quantification methods in diagnostic and foren-sic applications. Both have proven to be precise and robust. InqPCR, both parameters quantification cycle (Cq) and amplification
dx.doi.org/10.1016/j.bdq.2017.02.077



dx.doi.org/10.1016/j.bdq.2017.02.078



dx.doi.org/10.1016/j.bdq.2017.02.079



S lar Det

etptQgqtpt(duamgwaesaR

[

[

[

[

[

[

h

Das

JMC

1

U2

3

C4

PME

iidofptt


fficiency (AE) are estimated from the amplification curve datao quantify the DNA amount [1,2]. dPCR, which astonishinglyredates qPCR, allows copy number quantification based on parti-ioning a PCR bulk reaction into multiple smaller reaction entities.uantification is then achieved by assessing the distribution of tar-et molecules within the sample by Poisson statistics [3]. UnlikePCR, dPCR does not require a calibration curve for quantifica-ion. Moreover, according to recent reports, dPCR offers a morerecise and reproducible quantification as well as sensitive detec-ion of minority genetic targets. Numerous platform technologiese. g., droplet-based, chamber-based, microbead-based) have beeneveloped so far, however their accompanying dPCR software mod-les are exclusively tied to the platform and therefore lack generalpplicability [4]. As the analysis of the dPCR and qPCR data is stillatter of ongoing research, others and we have recently investi-

ated different software frameworks to fill this gap [5,6]. This talkill discuss novel and specific advances within the field of dPCR

nd qPCR data analysis, which have been made available to the sci-ntific community. The talk will focus on open source software andelected algorithms that can be used to build customized desktopnd remote browser applications.eferences

1] S. Pabinger, et al., A survey of tools for the analysis of quantitative PCR (qPCR)data, Biomol. Detect. Quantif. 1 (2014) 23–33, http://dx.doi.org/10.1016/j.bdq.2014.08.002.

2] A.-N. Spiess, et al., Impact of smoothing on parameter estimation inquantitative DNA amplification experiments, Clin. Chem. 61 (2015) 379–388,http://dx.doi.org/10.1373/clinchem.2014.230656.

3] M. Burdukiewicz, et al., Methods for comparing multiple digital PCRexperiments, Biomol. Detect. Quantif. 9 (2016) 14–19, http://dx.doi.org/10.1016/j.bdq.2016.06.004.

4] S. Rödiger, et al., R as an Environment for the Reproducible Analysis of DNAAmplification Experiments, R J. 7 (2015) 127–150,http://journal.r-project.org/archive/2015-1/RJ-2015-1.pdf.

5] A.-N. Spiess, et al., System-specific periodicity in quantitative real-timepolymerase chain reaction data questions threshold-based quantitation, Sci.Rep. 6 (2016) 38951, http://dx.doi.org/10.1038/srep38951.

6] S. Rödiger, et al., chipPCR: an R package to pre-process raw data ofamplification curves, Bioinformatics. 31 (2015) 2900–2902, http://dx.doi.org/10.1093/bioinformatics/btv205.


evelopment of an HRM-based tool for theutomated identification of nucleotideequences in large datasets

ean-Christophe Avarre 1,∗, Matthieu Vignoles 2,athieu Laffont 2, Lise Grewis 2,

hristelle Reynes 3,4

Institut des Sciences de l’Evolution de Montpellier,MR IRD-CNRS-UM-EPHE, Montpellier, FranceIDVet Genetics, Montpellier, FranceInstitute of Functional Genomics, UMRNRS-INSERM-UM, Montpellier, FranceLaboratory of Biostatistics, Informatics andharmaceutical Physics, UFR Pharmacy, University ofontpellier, France


Nucleic acid characterization by High Resolution Melting (HRM)s a simple, flexible, low-cost and powerful technique for identify-ng sequence variations, making it attractive for a broad range ofiagnostic and research applications including infectious diseases,ncology, epigenetics and even metabarcoding. Current procedures
or analyzing HRM curves mostly rely on unsupervised methods,rincipally via subtraction (difference) plots against a known con-rol sample, and less commonly on supervised methods throughhe use of discrimination analyses. If these procedures have proven

useful for discriminating a small number of variants, they are yetlimiting for analyzing large HRM data sets and do not provide pre-cise feedback to the user.

In this context, we have developed an innovative method thatenables the simultaneous discrimination of a large number ofvariants from their HRM profile. This method relies on the estab-lishment of a melting profile library, computes new descriptorsfrom the HRM curves for an optimal discrimination and offers afully automated analysis of melting profiles. The output consists inthe possibility to assign a given melting profile to an existing groupincluded in the reference library (assorted with a confidence index)or to reject any assignment in case of an unknown profile.

This method was first validated on a set of 19 nontuberculousmycobacterial species. Each species was represented by 3–20 bio-logical samples consisting of genomic DNA extracted either fromanimal tissues or from cultivated isolates. Each sample was ampli-fied with a unique pair of primers targeting the 16S-ITS region andyielding amplicons with sizes ranging from ∼230 to ∼350 bp. Melt-ing profiles of the corresponding amplicons were generated using3-9 replicates per sample. On a total of 95 samples, 91 were allot-ted to the right species. Automatic group rationalization led to splittwo species into two subgroups, suggesting that this method is ableto integrate intraspecific sequence variations.

The method was then applied to develop a diagnostic tool tar-geting five different pathogens responsible for abortive diseasesin cattle. Each pathogen was represented by 10 to 22 differentbiological samples consisting of genomic DNA extracted from dif-ferent matrices (e.g. feces, swabs, whole blood). Each biologicalsample was amplified and melted in 4 replicates with a ready-to-use mastermix containing 5 sets of primers, specific to the targetedpathogens. The limit of detection of this multiplex test was equiva-lent to that of the current individual tests using hydrolysis probes,around 10 copies/PCR. Moreover, all samples containing at least10 copies of pathogen(s) were correctly identified with high confi-dence.

These results underline the high potential of this novel HRM-based method for the simultaneous detection and identificationof a large number of nucleotide sequences, in both simplex andmultiplex formats.


A new paradigm in genetic data analysis on thethermo fisher cloud

Nivedita Majumdar ∗, Puneet Suri, Gloria Lam,David Woo, Shakila Pothini

Thermofisher Scientific, USAE-mail address:[email protected]

In this day and age, computation, storage and collation ofinformation, collation of analysis results from the large variety ofexperiments, are becoming vital to any biologically relevant discov-ery. This translates to millions of records in databases, requiringsophisticated algorithmic processing, cross-application analysis,interactive visualizations and infrastructure for collaboration. TheThermo Fisher Cloud platform offers such a solution with more thanten qPCR (quantitative polymerase chain reaction) and CE (capil-lary electrophoresis) apps for gene expression, genotyping, meltanalysis and Sanger sequencing. We are building an ecosystem to
eventually connect across all types of genetic analysis includingNGS (next generation sequencing) and MS (mass spectrometry) ona single platform, with a data or scientific question centric organi-zation of your information.
dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1016/j.bdq.2014.08.002

dx.doi.org/10.1373/clinchem.2014.230656









dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1016/j.bdq.2016.06.004

dx.doi.org/10.1038/srep38951







dx.doi.org/10.1093/bioinformatics/btv205








dx.doi.org/10.1016/j.bdq.2017.02.080



dx.doi.org/10.1016/j.bdq.2017.02.081



lar Det

faafictr

h

C(d

M

E

Ruhtgnltithdqtpa

ptaoaatpfCpttTaiptwsbttfatd


This talk will demonstrate the capabilities of our current plat-orm with a case study doing gene expression and genotypingnalysis. We will demonstrate the tools we provide for reviewingnd making sense of large volumes of data, with emphasis on ampli-cation curves. We will present tools such as the outlier wheel thatome together to enable the user to identify outliers, to identifyrends and patterns in their data, in order to make scientificallyelevant and reliable conclusions.


OMplementary Primer ASymmetric PCRCOMPASTM-PCR), a counter intuitive primeresign for PCR

arc Anglès d’Auriac

Norwegian Institute for Water Research, Norway-mail address: [email protected]

Since it was developed in the mid 80’, the Polymerase Chaineaction (PCR) has established itself as a central tool for molec-lar biology. Instrumentation, reagents as well as methodologyave regularly improved and expanded the capabilities of thisechnology. Limitations inherent to DNA and kit chemistries haveenerated guidelines for PCR-assay development. For instance,on-target amplification and in particular primer complementarity

eading to Primer Dimer formation is a well-known limiting fac-or to be reckoned with. Although various strategies have helpedmproved general robustness of PCR assays, primer complemen-arity usually is carefully avoided when designing PCR by using adoc software. Indeed, 3′-complementarity will extend the primersuring PCR elongation using one another as template, conse-uently disabling any possible further involvement in traditionalarget amplification. However, a 5′-complementarity will leave therimers unchanged during PCR cycles, albeit sequestered to onenother, therefore also competing with target amplification.

In this work we show that 5′-complementarity betweenrimers may be exploited in a new asymmetric PCR method,he COMplementary-Primer-Asymmetric (COMPASTM)-PCR, tochieve effective double strand target PCR amplification. More-ver, such a design may paradoxically reduce spurious non-targetmplification by actively sequestering the limiting primer. Usingsymmetric primer concentration is not a new approach but has,o our knowledge, only been previously used for enhancing theroduction of single strand amplicon from a target sequence, i.e.or probe detection assays. In the presence of the target sequence,OMPAS-PCR initiates target linear amplification with the excessrimer, hence progressively changing the stoichiometry of the reac-ion so that priming of the limiting primer to the target strand andarget extension of the complementary strand is gradually favored.his general principle was developed using 5S rDNA direct repeatss target sequences to design a species-specific assay for identify-ng Salmo salar and Salmo trutta using almost fully complementaryrimers overlapping the same target sequence. This initial applica-ion, published in PLoS One (doi: 10.1371/journal.pone.0165468),as designed so that both primers are complementary to the

ame genomic sequence target. This approach may in principlee applied to any tandem direct repeats DNA motifs of interest asarget sequences. Ribosomal genes and in particular the 5S rDNAandem direct repeats are found in all eukaryotic cells and are there-ore suitable for developing new specific complementary-primerssays. We believe that further understanding and modelling of
hese COMPAS-PCR principles could be incorporated in primeresign software.

This small paradigm shift, using highly complementary primersfor PCR, should help develop or improve PCR assays, increasingdesign possibilities available to the molecular scientist.


Comparative procedures for sample processingand development of qPCR assays for the rapiddetection of grapevine and citrus pathogens

Emir Hodzic

School of Veterinary Medicine, University ofCalifornia at Davis, United StatesE-mail address: [email protected]

Different instruments and methods were used for tissue homog-enization, RNA extraction and qPCR based detection of grapevineand citrus RNA viruses were evaluated. Semi-automated and auto-mated homogenization techniques were compared to processsamples from grapevine and citrus. Four different high throughputautomated nucleic acid extraction platforms were compared withthe RNeasy plant extraction kit for their capacity and efficiency ofextracting viral RNA from grapevine and citrus infected tissues. TheRNA prepared from each extraction platform was then used as tem-plate for a comparative analysis. The study showed that a thoroughhomogenization of grapevine and citrus tissues using the TissueLyser as well as DNase digestion of the purified RNA prior to cDNAsynthesis improved the virus detection and yielded the lowest Cqvalues in RT-qPCR. Comparison of different RNA extraction meth-ods showed that methods implementing the magnetic bead-basedtechnology were superior to other methods used.

The development of diagnostic systems to effectively detectmultiple targets in a single assay has provided a constant techno-logical challenge. The main goal of this study was to develop a rapid,sensitive and specific multiplex RT-qPCR assays for the detectionand quantification of viruses in grapevines and citrus that can beincorporated into routine virus detection protocols. The proposedapproach can then be used as a robust diagnostic tool for grapevineand citrus germplasm screening programs and for the certificationprogram to ensure clean plant material for propagation.

A single real-time multiplex qPCR assay for the simultaneousdetection of Grapevine virus A, B and D (GVA, GVB and GVD)was developed, using three different fluorescently labeled minorgroove binding probes. This multiplex RT-qPCR was compared tosingleplex RT-qPCR designed specifically for each virus and a con-ventional multiplex RT-PCR. The results showed that the developedmultiplex RT-qPCR assay was a cost-effective diagnostic tool thatcould streamline the testing of grapevine viruses, and replace thesingleplex RT-qPCR assays, thus reducing time and labor whileretaining the same sensitivity and specificity.

For detection of Citrus tristeza virus (CTV), Citrus psorosis virus(CPsV), and Citrus leaf blotch virus (CLBV) a single multiplex RT-qPCRwas developed and validated using the same approach as above. Toincrease the detection reliability, coat protein from large number ofdifferent isolates of CTV, CPsV and CLBV were sequenced and multi-ple sequence alignments were generated. No significant differencein detection limits was found and specificity was not affected bythe inclusion of the three assays in a multiplex RT-qPCR reaction.

Adopting compatible multiplex RT-qPCR testing protocols forgrapevine and citrus viruses as well as other RNA and DNAregulated pathogens will provide a valuable alternative tool forpathogen detection and efficient program implementation.


dx.doi.org/10.1016/j.bdq.2017.02.082



dx.doi.org/10.1016/j.bdq.2017.02.083



dx.doi.org/10.1016/j.bdq.2017.02.084

S lar Det

S

T

L

S

SS

Cpt

F

Ef

tdsal–

adcktdsDeatis

t

h

Ha

SI

E

idydlIsoccy


ession MG – MicroGenomics & Single-Cell-qPCR

ime: Wednesday, 05/Apr/2017: 2:00 pm – 4:30 pm


ession Chair: Mikael Kubista, TATAA Biocenter AB, Sweden

ession Chair: Anders Ståhlberg, University of Gothenburg,weden

omputational analysis of single-cell RNA-seqrofiles identifies lineage choice and gradedransitions in myeloid progenitors

abian J. Theis

Helmholtz Zentrum München/TUM, Germany-mail address:[email protected]

Single-cell technologies have gained popularity in developmen-al biology because they allow resolving potential heterogeneitiesue to asynchronicity of differentiating cells. Common data analy-is encompasses normalization, followed by dimension reductionnd clustering to identify subgroups. However, in the case of cellu-ar differentiation, we may not expect clear clusters to be presentinstead cells tend to follow continuous branching lineages.

We show that modeling the high-dimensional state space asdiffusion process, where cells move to close-by cells with a

istance-dependent probability well reflects the differentiatingharacteristics. Based on the underlying diffusion map transitionernel, we then order cells according to a diffusion pseudo-ime (DPT), which allows for a robust identification of branchingecisions and corresponding trajectories of single cells. We demon-trate the method on scRNA-seq data of myeloid differentiation.PT identifies a dominant branching into different myeloid lin-ages and a minor subpopulation of lymphoid outliers. Moreover,graded transition reflecting erythroid differentiation is identified

hat dissent from previously stated cluster sequences. We finallydentify driver genes and propose how to include additional dataets for integrative analysis across multiple downstream lineages.

Altogether, this illustrates that the concept of discrete transi-ions of progenitors to developed cells may need to be adapted.


igh resolution single cell analysis in complexdult tissues

tefan Günther ∗, Michail Yekelchyk,sabelle Salwig, Jens Preussner, Thomas Braun

MPI for Heart and Lung Research, Germany-mail address: [email protected]

Molecular analysis of complex tissues in adult model organismss a common tool for deciphering processes and networks duringevelopment, aging and disease. The majority of such “omic” anal-ses were done using whole tissue/organs or in macro or microissected anatomical regions. However, the complexity and cellu-

ar composition of such samples prevent high-resolution analysis.dentification of small subpopulations or specific changes withinmall cell populations are usually not detected due to massiveverrepresentation of signals/data points from bulk cells. To over-
ome these problems and to gain insights into subpopulation ofells many attempts are being made to switch from bulk anal-ses to identification of molecular changes in single cells. This

approach allows a very high-resolution analysis, but is associ-ated with numerous other problems that have to be resolved toachieve meaningful insights. Our main focus is on the transcrip-tomics of individual cells and subpopulations. Many techniquesand tools were developed during last years to obtain transcrip-tomics data from hundreds or thousands of cells from differentmodel organisms, organs and tissues. Nonetheless, many exper-iments require special tissue-dependent conditions limiting theuse of standard methods. Here, we show data from 2 projects,which faces special challenges regarding cell size and number ofcellular subpopulations within individual samples. In collabora-tion with Wafergen/Takara Bio we demonstrate that the ICELL8TM

Single-Cell System can be used to obtain high-resolution single celldata for particularly large cells or complex cell populations, whichare difficult to analyze with other techniques. Our results providenew insights into the heterogeneity of cell populations and thetranscriptional networks that regulate biological processes in thecorresponding tissues.


A cost effective 5′ selective single celltranscriptome profiling approach

Pascal Barbry 1,2,∗, Marie-Jeanne Arguel 1,2,Kévin Lebrigand 1,2, Agnès Paquet 1,2,Sandra Ruiz-Garcia 1,2,Laure-Emmanuelle Zaragosi 1,2,Rainer Waldmann 1,2

1 Université Côte d’Azur, France2 CNRS, FranceE-mail address: [email protected]

Single cell RNA sequencing approaches are instrumental in stud-ies of cell-to-cell variability. 5′ selective transcriptome profilingapproaches allow simultaneous definition of the transcription startsize and have advantages over 3′ selective approaches which justprovide internal sequences close to the 3′ end. The only currentlyexisting 5′ selective approach requires costly and labor intensivefragmentation and cell barcoding after cDNA amplification. Wedeveloped an optimized 5′ selective workflow where all the cellindexing is done prior to fragmentation. With our protocol, cellindexing can be performed in the Fluidigm C1 microfluidic device,resulting in a significant reduction of cost and labor. We alsodesigned optimized unique molecular identifiers that show lesssequence bias and vulnerability towards sequencing errors result-ing in an improved accuracy of molecule counting. We providecomprehensive experimental workflows for Illumina and Ion Pro-ton sequencers that allow single cell sequencing in a cost rangecomparable to qPCR assays.


Deeper understanding starts with a single cell

Afif M. Abdel Nour 1,∗, Georges Nemer 2,Lara Hanna Wakim 1, Esam Azhar 3,4

1 Faculty of Agricultural and Food Sciences, HolySpirit University of Kaslik, Jounieh, Lebanon2 Department of Biochemistry and MolecularGenetics, American University of Beirut-Medical
Center, Beirut, Lebanon3 Special Infectious Agents Unit-Biosafety Level 3,King Fahd Medical Research Center, King AbdulazizUniversity, Jeddah, Saudi Arabia


dx.doi.org/10.1016/j.bdq.2017.02.085



dx.doi.org/10.1016/j.bdq.2017.02.086



dx.doi.org/10.1016/j.bdq.2017.02.087


lar Det

4

FUE

eiOptfqsdfiqmcctTtievia

h

Wt

RR

E

ohaialttMmavmflt

h

the suitability of the SMARTer approach for applications such asbiomarker discovery.



Medical Laboratory Technology Department,aculty of Applied Medical Sciences, King Abdulazizniversity, Jeddah, Saudi Arabia-mail address: [email protected]

In the past few years Single cell research had tremendous inter-st from public and private researchers and as a result manynstitutions were striving in publishing single cell related papers.ver the course of the last 10 years the growth rate of publishedapers was not far from double digits. Although, most of the pro-ocols are based on qPCR, we are presenting today our “one stopull solution” for needs in single cell analysis this including thePCR as well as the ddPCR. This workflow includes rapid, ultra-ensitive analysis of 2-10 genes in as low as single cell. We haveeveloped a cost effective and streamlined gene expression pro-ling workflow that enables monitoring of hESC/iPSC culture foruality control, reprogramming, and lineage determination withinimal sample consumption. The 4-step integrated workflow

ouples column-free total RNA isolation (gDNA-free) with lysate-ompatible reverse transcription and target specific (up to 100)andem pre-amplification followed by SYBR-based qPCR analysis.he entire workflow can be completed from sample to quantita-ion of up to 100 targets in less than 1 day. The STDEV from threendependent pre-amplifications reactions is only 0.09, and 97% ofxpressed targets show less than 0.75 deviation from predictedalues. Other tools are also presented including single cell sequenc-ng. The reductionist approach to study organisms using single cellnalysis proved to be efficient specially in oncology.


hole transcriptome subcellular profiling ofhe Xenopus oocyte

adek Sindelka ∗, Pavel Abaffy, Silvie Tomankova,avindra Naraine, Mikael Kubista

Institute of Biotechnology-Biocev, Czech Republic-mail address: [email protected]

Starting from a single fertilized oocyte, through manifoldf divisions a complex organism is developed that has distinctead-tail (bottom-up), left-right and dorsal-ventral (back-belly)symmetries. One of the main challenges in developmental biologys to understand how and when these asymmetries are generatednd how they are controlled. The African clawed frog (Xenopusaevis) is an ideal model for studies of early development thanks toheir very large oocytes. We have developed a unique molecularomography platform based on qRT-PCR, RNA-seq and UPLC-ESI-

S/MS to measure asymmetric localization of fate determiningRNAs, non-coding RNAs and proteins within the oocyte and

mong the early stage blastomeres. The first axis called animal-egetal, is formed during oogenesis and we found mRNA andicroRNA gradients determining its formation. First cell division

ollowing fertilization producing 2-cell stage embryo forms the
eft-right, and second cleavage generating 4-cell embryos specifieshe dorsal-ventral axis.


Postersessions:

Time: Monday, 03/Apr/2017: 6:00 pm – 10:00 pm

Location: Foyer – lower level, Lunch and Poster Session

All posters will be displayed in all three poster sessions (PO1, PO2and PO3). Poster setup is on Monday afternoon and they areavailable until Wednesday afternoon.

PO 01Ligation-free small RNA sequencing librariesfrom clinical samples using SMART

®technology

Sara Gonzalez-Hilarion 1,∗, Nathalie Bolduc 2,Simon Lee 2, Rachel Fisch 2,Kahraman Tanriverdi 3, Evangelia Malahias 3,Ekaterina Mikhalev 3, Jane Freedman 3,Andrew Farmer 2

1 Takara Bio Europe, France2 Takara Bio, USA3 University of Massachusetts Medical School,Department of Medicine, Division of CardiovascularMedicine, USAE-mail address:sara [email protected]

Small non-coding RNAs regulate gene expression via diversemechanisms and facilitate fundamental cellular processes such astranscript splicing and protein translation. Moreover, small RNAsmay act at sites different from their original production via incorpo-ration and transport in bodily fluids. Obtaining an accurate portraitof small RNA expression levels from small sample inputs carriespotential both for the fulfillment of basic research objectives andthe development of novel therapeutics and clinical diagnostic solu-tions. Towards this end, we have developed a novel, ligation-freeapproach for the preparation of small RNA sequencing librariesthat leverages 3′ RNA polyadenylation followed by cDNA synthesisand template switching. This approach minimizes sample repre-sentation bias and is sensitive enough to accommodate inputs of aslittle as 1 ng of total RNA. Here we present data demonstrating theaccuracy, sensitivity, and reproducibility afforded by the SMARTer

®

smRNA-Seq Kit for Illumina®

. Using a library of synthetic miRNAs,we show that the SMARTer method generates a more accurate rep-resentation than the typical adapter ligation method. Furthermore,we successfully created high-quality libraries using 400 pg to 1 ngof RNA extracted from plasma and serum samples, demonstrating


dx.doi.org/10.1016/j.bdq.2017.02.088



dx.doi.org/10.1016/j.bdq.2017.02.089



dx.doi.org/10.1016/j.bdq.2017.02.090

S lar Det

PCN

EYCASJW

E

sDomdrp

hoPisepsDfyaCevp

iotqii

h

PGc

T

E

dSggico


O 02omplete “Sample-to-Result” highly automatedGS and qPCR workflow for clinical diagnostics

lian Rakhmanaliev ∗, Tatiana Ivanova,in Kum Ng, Alex Yeo, Harry Suhardi,aroline Villy, Mei Qi Yee, Kevin Tan,manda Fan, Andy Soh, Pramila Ariyaratne,i Kee Tan, Jocelyn Wong, Jiang Xia, Grace Tan,asmin Ang, John Ong, Charlie Lee, Gerd Michel,

en Huang

Vela Diagnostics Pte. Ltd., Singapore-mail address: [email protected]

Sanger sequencing and real-time PCR methods have been thetandard molecular methods in clinical diagnostics for decades.eep sequencing or Next Generation Sequencing (NGS) technol-gy revolutionized the field of genomics, transcriptomics andetagenomics and is now rapidly becoming a routine method in

ifferent areas of clinical diagnostics such virology, oncology, drug-esistance monitoring, non-invasive prenatal tests, microbiology,recision medicine, etc.

Vela Diagnostics developed complete (“Sample-to-Result”)ighly automated multi-purpose Sentosa workflow, which consistsf: (1) a robotic liquid handling system for nucleic acid extraction,CR set-up and/or NGS library preparation (Sentosa SX101); (2)nstruments for real-time PCR or template preparation and deepequencing (Ion Torrent’s PGM system); (3) kits for nucleic acidxtraction, target specific real-time PCR-based tests, NGS libraryreparation assays and reagents for deep sequencing; (4) assaypecific applications, and (5) data analysis and reporting software.ifferent diagnostic applications employ the same robotic platform

or qPCR set-up and preparation of NGS libraries. In less than 5ears Vela Diagnostics developed 7 NGS-based viral and oncologyssays and more than 20 qPCR-based viral, microbial and oncologyE-IVD tests, which can be run on the same system. In addition, sev-ral extraction kits were developed to isolate nucleic acids fromarious types of clinical samples, including FFPE, whole blood,lasma/serum, swabs, sputum, stool and urine.

Sentosa qPCR and NGS workflow appears as a robust and efficientn vitro diagnostics (IVD) tool for the detection and/or quantitationf a wide range of bacterial and viral pathogens as well as muta-ions in human genes. Such comprehensive combined NGS andPCR workflow that can help fill some existing gaps in the qual-

ty of molecular diagnostics will take the necessary steps towardsmproving patient outcomes.


O 03ene expression variance as a proxy forhronological age

ong Shu Li ∗, Michael Petrascheck, Andrew I. Su

The Scripps Research Institute, United States-mail address: [email protected]

Aging, previously thought to be a random process of entropicecay in living organisms, is now known to be genetically regulated.ince the discovery that inhibiting insulin/IGF-1 signaling in C. ele-ans extends lifespan, much work has focused on finding additional
enetic modulators of lifespan. Here we present “drift” as a geneticndicator of chronological age which depends upon the harmonicorrelated expression of gene sets. Drift is defined as the variancef the relative expression values of a group of genes with respect

to themselves at an earlier time. As genes need to work in con-cert with other genes, we hypothesized that increasing differencesin the ratio of gene products within biological pathways would bedetrimental to an organism’s health and contribute to aging. Wetherefore sought to develop a genetic biomarker for chronologicalage which would be useful for interrogating an organism’s expectedchronological age without having to wait for the organism to per-ish. In addition, we aimed to use drift to uncover specific areas ofbiology which are critical to lifespan extension.

Gene expression values of C. elegans cohorts were measured viaRNA-seq (Illumina). Mianserin, a small-molecule antidepressant,was known from previous studies to increase mean C. elegans life-span and was used to generate long-lived N2 worms in the study.Two cohorts of worms were generated. Worms were fed either sol-vent or 50 �M mianserin on day one of adulthood, and RNA washarvested for sequencing on days 1, 3, 5, and 10 of adulthood.Genomes were aligned to the reference genome from WormBase.Data analysis was performed in Python and R.

Drift analysis was performed for 10,879 C. elegans genes at atranscriptome-wide level. While 19,196 genes were sequenced,some genes had to be removed from the analysis due toundetectable expression values, which interfered with the nor-malization procedure for calculating drift. For the 10,879 analyzedgenes, we found that their drift values increased log-linearly withchronological age, and was consistently reduced across the entireworm’s lifespan when exposed to mianserin. This supports ourprevious findings that mianserin specifically increases the youngperiod of a worm’s life.

We also examined how well genes grouped together by GeneOntology (GO) terms drifted in order to identify biological areaswhich may be significant modulators of C. elegans lifespan. Sincewe found that the entire transcriptome exhibited drift behavior, wecompared GO term gene sets to size-matched gene sets randomlysampled from the population of all genes. This allowed us to moreaccurately estimate how well a GO term grouped genes of simi-lar biological function together. We identified bacterial responsegenes, protein kinase activity regulators, and immune responsegenes to be most highly correlated with drift. In conclusion, wehave created a molecular proxy correlating with chronological agewhich is sensitive to changes in worm lifespan.


PO 04Efficient isolation on Vero.DogSLAMtag cell lineand whole genome characterization by nextgeneration sequencing of dolphin morbillivirus

Simone Peletto 1,∗, Claudio Caruso 1,Cerutti Francesco 1, Modesto Paola 1,Cristina Biolatti 1, Alessandra Pautasso 1,Carla Grattarola 1, Federica Giorda 1,Sandro Mazzariol 2, Walter Mignone 1,Loretta Masoero 1, Cristina Casalone 1,Pier Luigi Acutis 1

1 Istituto Zooprofilattico Sperimentale del Piemonte,Liguria e Valle d’Aosta, Turin, Italy2 University of Padova, Padua, ItalyE-mail address: [email protected]

Dolphin morbillivirus (DMV) is regarded as an emerging threat
being the cause of recent epidemics worldwide. In 1990–1992,a DMV outbreak in the Mediterranean Sea involved thousandsof striped dolphins with high mortality. Another major epi-demic event was the DMV outbreak in 2006–2008 affecting three


dx.doi.org/10.1016/j.bdq.2017.02.091



dx.doi.org/10.1016/j.bdq.2017.02.092



lar Det

cf(tsrDao

rwLRgvu

dlctrdltos

scoofpstcn2Igit

h

PDid

ABCGM

1

a2

G3

DT


etacean species along the Mediterranean coast. The DMV genomerom the 1990–1992 epidemics is the only cetacean morbillivirusMV) genome that has been completely sequenced. We reporthe first retrospective isolation of DMV from a striped dolphintranded along the Italian coast in 2008. This viral isolate, rep-esentative of the 2006–2008 Mediterranean epidemic (hereafterMV IZSPLV 2008), efficiently growth on Vero.DogSLAMtag cellsnd was submitted to whole genome sequencing by NGS technol-gy.

The striped dolphin was an adult female stranded along the Ligu-ian coast in October 2008. In 2015, the brain of the striped dolphinas processed for virus isolation using Vero.DogSLAMtag cell line.

ibraries were submitted to NGS on an Illumina MiSeq platform.eads passing the quality filter were aligned to the DMV referenceenome (NC 005283) using the BWA software. Read mapping wasisualized with Tablet to obtain a consensus genome. MEGA7 wassed for maximum likelihood (ML) phylogeny.

Successful viral isolation on Vero.DogSLAMtag cell line wasemonstrated by CPE observed after 5 days. The final genome

ength obtained from the read assembly was 15,667 nucleotidesovering 99.78% of the DMV reference genome. Comparison ofhe DMV IZSPLV 2008 and the 1990–1992 DMV strain sequencesevealed 157 nucleotide mutations and 47 amino acid changes atifferent sites. The sequence similarity was 98.7% at the full genome

evel. Additionally, the DMV IZSPLV 2008 isolate was aligned withwo partial sequences identified in Spain during the 2006–2008utbreak in a striped dolphin and a pilot whale; in this case,equence similarities were 99.8% and 99.9%, respectively.

In MV diagnostics, viral isolation is still considered the “goldtandard”, even though efficient propagation of live virus is diffi-ult to achieve. Our results seem to confirm an increased sensitivityf Vero.DogSLAMtag cells for isolating cetacean MV. The successfuln-culture replication of the virus allowed performing NGS directlyrom the viral RNA, without prior PCR amplification. We thereforerovide the scientific community a second DMV genome repre-entative of another major outbreak, the 2006–2008 epidemics inhe Mediterranean Sea. Analysis of nucleotide sequence similarityonfirmed that the two viruses are highly correlated. Phyloge-etic analysis suggests that the DMV strain circulating during the006–2008 epidemics emerged from the 1990–1992 DMV strain.

nterestingly, genome comparison revealed that the neglected Lene, although encompassing ∼40% of the DMV genome, indeedncludes 74% (35 out of 47) of the diversity (amino acid changes)hat differentiate the strains of the two epidemics.


O 05eveloping a potential biomarker set of miRNAs

n packed RBCs to detect autologous bloodoping

nna Haberberger 1,∗, Veronika Mussack 1,enedikt Kirchner 1, Irmgard Riedmaier 1,2,hristian Wichmann 3, Raymund Buhmann 3,eorg Wittmann 3, Reinhard Henschler 3,4,ichael W. Pfaffl 1

Technical University Munich, Animal Physiologynd Immunology, Freising, GermanyEurofins Medigenomix Forensik GmbH, Ebersberg,ermany
Ludwig-Maximilians University Hospital,epartment of Transfusion Medicine, Cellherapeutics and Hemostaseology, Munich, Germany

4 Blutspende Zürich, Medizinischer Dienst, Zurich,SwitzerlandE-mail address: [email protected]

Autologous blood doping provides an athlete with an increasedred blood cell (RBC) mass after transfusion of his own packed RBCs,which results in enhanced performance as compared to a non-doped athlete. Nevertheless, it is also associated with serious healththreats like stroke or pulmonary embolism. Although this kind ofdoping is prohibited by the WADA (World Anti-Doping Agency)since 1986 there is still no reliable test to detect this kind of doping.The hypothesis was that the miRNA signature of packed RBCs dif-fer from fresh whole blood due to blood processing, buffer, storagetemperature, and storage time. In this study, concentration wasdirected only on the increased miRNAs of stored RBC units on theaspect that only increased miRNA levels could be measured aftertransfusion.

To verify this hypothesis, three venous blood samples weretaken from 12 healthy and athletic subjects before blood dona-tion and from their blood bags at several time points during bloodstorage. Here, all 12 subjects were investigated using small RNASequencing, and analyzed using the DESeq2 algorithm.

The resulting data shows a significant increase in more than120 miRNAs in the packed RBCs compared to fresh whole blood,which is due to preservative solutions and processing. A list of theTop 20 increased miRNAs without natural variation and with thegreatest p-values was drawn up. This list distinguishes betweensamples freshly collected from subjects and their packed RBCs afterprocessing.

The next step will be the discovery of changes during storageuntil week 6 to adjust the preliminary miRNA signature accordingto these storage changes und to test this potential miRNA signaturein an autologous blood doping study.


PO 06Different isolation strategies of exosomalextraction kits have a major influence on smallRNA capturing and expression profiles inclinical settings

Benedikt Kirchner 1,2,∗, Dominik Buschmann 1,3,Stefan Kotschote 4, Michael Bonin 4,Marlene Reithmair 3, Gustav Schelling 5,Michael W. Pfaffl 1

1 School of Life Sciences Weihenstephan, TechnicalUniversity Munich, Germany2 Dr. von Hauner Childrens’s Hospital, LudwigMaximillian Universität, Munich, Germany3 Institute of Human Genetics,Ludwig-Maximilians-University Munich, Germany4 IMGM Laboratories GmbH, Planegg, Germany5 Department of Anesthesiology,Ludwig-Maximilians-University Munich, GermanyE-mail address: [email protected]

Extracellular vesicles in general and exosomes in particu-lar have become an important object of research especially inbiomarker development, due to the special role they and theircargo play in cell-to-cell communications. Since isolation via differ-ential ultracentrifugation is time-consuming and labor intensive,
commercially available extraction kits take advantage of var-ious biochemical and physical properties to isolate exosomesfrom biofluids or cell-culture supernatant. Although all isolation
dx.doi.org/10.1016/j.bdq.2017.02.093



dx.doi.org/10.1016/j.bdq.2017.02.094



S lar Det

scatr

iccusksei

loSodsae

fhtac

h

PNP

LBS

1

2

E

smLlpwtpteTmtoflunAd


trategies (e.g. membrane affinity, precipitation, size exclusionhromatography) offer their own advantages and disadvantagesthorough evaluation on their impact on downstream applica-

ions like NGS is still missing, especially in the context of clinicallyelevant settings or samples like liquid biopsies.

Extracellular vesicles were isolated from 1 ml serum of healthyndividuals and critically ill patients (n = 10 each) using 4 differentommercially available isolation kit alongside differential ultra-entrifugation (n = 8). Total RNA yield and integrity were evaluatedsing capillary gel electrophoresis and holistic small RNA expres-ion profiles were generated by NGS. Extracellular vesicle isolationit-specific influences were assessed by comparing library size,equence length distribution, unsupervised clustering and differ-ntial expression analysis between sample matrices as well assolation strategies.

Total RNA yield differed greatly (p-value = 0.002) between iso-ation strategies with precipitation (4505 ± 3329 pg/�l) greatlyutperforming size-exclusion chromatography (157 ± 197 pg/�l).ampling from critically ill patients reduced RNA yield for all meth-ds by a factor of 1.5–3.8 (p-value = 0.002). Even more strikingifferences were revealed by small RNA NGS. Although all isolationtrategies were able to distinguish between samples from healthynd critically ill individuals to a certain degree, mapped miRNAxpression profiles varied significantly.

A major impact on small RNA expression profiles could be shownor all EV isolation kits and strategies, respectively. Our findingsighlight the importance of further optimization and standardiza-ion of exosomal isolation methods in differing sample matricesnd special attention needs to be paid to obtain reproducible andomparable biomarker signatures from liquid biopsies.


O 07EXTERA XT DNA Library Preparation onIPETMAX

aura B. Simdon 1, Seth A. Hanson 1,randon Converse 2, Brendan Keogh 2,cott Monsma 2, Tristan J. Berto 1,∗

Gilson Inc., United StatesLucigen Corp., United States-mail address: [email protected]

Reliable and reproducible library preparation is critical foruccessful next generation sequencing. In this project we auto-ated the liquid handling steps of the Illumina Nextera XT DNA

ibrary Preparation Kit workflow and then constructed replicateibraries using either a benchtop liquid handler or manual pipettingerformed by an experienced technician. Five automated scriptsere developed, corresponding to the Tagmentation, Amplifica-

ion, Library Cleanup, Library Normalization, and Library Poolingortions of the workflow. The PIPETMAX was equipped withwo motorized, multichannel air displacement pipette heads thatnable precise single or multichannel pipetting from 1 �L to 200 �L.he compact benchtop instrument permits up to nine bed ele-ents to be employed during an automated script, including

ips, microplates, tube racks, and accessories such as an on-bedrbital shaker. A custom rack was constructed to hold SLAS-ootprint labware in the portrait orientation (as opposed to theandscape orientation on a standard PIPETMAX tray) and was
sed in the Amplification plate setup script to enable multichan-el pipetting of Illumina TruSeq primers into the proper matrix.s part of the Library Cleanup and Library Normalization proce-ures, the Agencourt
®AMPure

®XP PCR purification system was


automated using an on-bed magnetic bead separator rack. ThePIPETMAX instrument can carry out all steps of the AMPure XP PCRcleanup procedure without manual intervention, including rais-ing and lowering the magnets, resuspending paramagnetic beads,and transferring all liquids such as sample, wash solution, 80%ethanol and resuspension buffer. The automated AMPure XP scriptalso allows the end user to modify several variables such as num-ber of samples to process, sample volume, bead volume, numberof wash steps, and incubation times. The wizard-style TRILUTIONmicro software interface provides real-time updates on the runstatus and produces run reports that help reduce errors associ-ated with manual data entry. To assess the library quality for NGSlibraries constructed using the automated procedure vs. manualliquid handling, twelve Nextera XT DNA libraries were constructedusing PIPETMAX and an additional twelve were prepared usingmanual pipetting. Each library was prepared from 1 ng of E. coligenomic DNA. Library size was assessed using an Agilent BioAn-alyzer. All 24 libraries were pooled and sequenced in one laneof an Illumina MiSeq instrument. Each library was downsampledto 312,500 reads, which yielded >8× coverage for all 24 libraries.Both library preparation methods generated high quality data with>95% mapped reads and optimal quality scores. The percentageof mapped reads was almost identical for libraries prepared withautomated liquid handling or manual liquid handling. The librariesprepared with PIPETMAX exhibited smaller standard deviation andvariance, consistent with the reproducibility of liquid handling onthis system.


PO 08SiMSen-Seq: Simple, multiplexed, PCR-basedbarcoding of DNA for sensitive mutationdetection using sequencing

Stefan Filges 1,∗, Gustav Johansson 1,Paul Krzyzanowski 2, Tony E. Godfrey 3,Anders Ståhlberg 1

1 Department of Pathology, Sahlgrenska CancerCenter, Institute of Biomedicine, SahlgrenskaAcademy at University of Gothenburg,Medicinaregatan 1F, 405 30 Gothenburg, Sweden2 Ontario Institute for Cancer Research, MaRS Centre,661 University Avenue, Suite 510, Toronto, Ontario,Canada M5G 0A33 Department of Surgery, Boston University School ofMedicine, 700 Albany Street, Boston, MA 02118, USAE-mail address: [email protected]

The detection of extremely rare variant alleles is becom-ing increasingly relevant in many areas of clinical and basicresearch, such as oncology, prenatal diagnostics and transplanta-tion medicine. Introducing molecular barcodes during NGS librarypreparation to uniquely tag target DNA allows the subsequentremoval of polymerase errors incurred during library generationand sequencing. The SiMSen-seq protocol allows flexible, easy gen-eration of barcoded libraries with minimal DNA input using tworounds of PCR and enables the detection of rare variants below 0.1%allele frequency. While the generation of libraries using barcodedPCR primers is an elegant, simple and fast solution, the detectionof rare variants from complex matrices such as human plasma or
serum necessitates highly optimized PCR assays and conditions.Here, we present major improvements to the original SiMSen-seq protocol. Through assay optimization and enhanced workflowsthe dynamic range and sensitivity to detect rare mutations are
dx.doi.org/10.1016/j.bdq.2017.02.095



dx.doi.org/10.1016/j.bdq.2017.02.096



lar Det

isaafwaha

h

PTwa

HM

Eh

sNhop

mTresatTNsssoi

lwtthmprsa

c


ncreased, while losses of target molecules are minimized at theame time. We outline suggestions for assay development to gener-te highly sensitive and specific assays to maximize library qualitynd recovery of target molecules. We also developed a softwareor de-barcoding and error correction from raw sequencing data,hich delivers detailed information on molecule counts and vari-

nt detection. Thus, the optimized SiMSen-seq workflow facilitatesighly accurate variant detection in challenging sample types suchs liquid biopsies.


O 09empO-Seq: High-throughput surrogate andhole transcriptome targeted gene expression

nalysis

arper VanSteenhouse ∗, Pete Shepard,ilos Babic, Joanne Yeakley, Bruce Seligmann

BioSpyder Technologies, United States-mail address:[email protected]

TempO-Seq®

is a novel highly multiplexed targeted RNA expres-ion profiling assay that makes extremely efficient use of existingGS platforms for readout. Its unique attributes enable manyigh-throughput applications where RNA-Seq or other meth-ds are too limited in performance or throughput, or are cost-rohibitive.

Sample input does not require RNA extraction/isolation, anday be lysates from cells or tissue, including FFPE, or purified RNA.

he assay is robust to degradation or fixation, generating equivalentesults even with these challenging samples. TempO-Seq achievesxceptionally high performance (reproducible differential expres-ion, single-base selectivity, and single-cell sensitivity) by utilizingnnealing and ligation steps without the limitations of reverseranscription or pre-amp using a simple add-only assay protocol.empO-Seq generates a reduced complexity barcoded and pooledGS library, is robust to sequencing errors, and minimizes neces-

ary bioinformatics, supporting very high throughputs with 384+amples multiplexed per flowcell lane. The capture-free assay isimple to perform manually in 96-well format with 1 h hands-n using common laboratory equipment including any PCR/qPCRnstrument, and may be automated.

Most importantly, the assay provides precise data withog2 R2 > 0.99, average CVs < 15%, and essentially no background,

hich permits even small differences down to 1.2-fold (padj < 0.05)o be accurately measured without interference from false posi-ives and enables comparison between disease states, intercellulareterogeneity, or compound dose-response effects. Probe perfor-ance is independent of context within different pools, allowing

anels to be customized without painstaking re-optimization whileetaining the ability to compare results. Pre-validated assays can beupplemented with custom-designed isoforms, cSNPs, splice vari-nts, fusion genes or other mutations.

We demonstrate TempO-Seq performance and cross-platformomparisons in a number of applications, including:

(i) kits measuring ∼2700 genes representing a human surrogate
transcriptome assay (with which >50 samples can be run on asingle MiSeq flowcell),
(ii) the human whole transcriptome assay against 38,247 RefSeqIDs,


(iii) single-cell analysis,(iv) degraded RNA and FFPE samples, and(v) non-human model species.


PO 10SIRV SUITE: RNA-Seq quality metrics andcomparison platform

Igor Holländer, Patrick Schagerl, Lukas Paul ∗,Torsten Reda

Lexogen GmbH, AustriaE-mail address: [email protected]

Despite significant advances in transcriptome sequencing themajority of samples is processed without external standards. Thishinders flawless comparisons between experiments and preventswhole RNA sequencing (RNA-Seq) to reach the next level and todevelop into standardized analytical tools for clinical applications.Lexogen, a specialized transcriptomics company, addresses thisproblem by providing Spike-In RNA Variants (SIRVs) which con-dense the complexity of transcriptomes in a nutshell. Tiny amountsof SIRVs processed as part of the RNA samples allow for the evalu-ation and monitoring of RNA sequencing with respect to transcriptisoform detection and quantification. However, because standardsrequire standardized data processing Lexogen presents the SIRVSUITE, a new software package for SIRV data evaluation. It is easyto operate and can be integrated in every RNA sequencing pipeline.The SIRV SUITE computes the main quality metrics of gene expres-sion measurements like coefficient of deviation, precision, accuracyand differential accuracy. The results are presented in overviewfigures, summarized in quality control forms, and saved in a database. The linked metadata and “RNA-Seq fingerprint” SIRV qual-ity metrics allow to systematically calculate the concordance ofRNA sequencing experiments. Data can be compared to referenceson the bases of meaningful autonomous control subsets. The SIRVSUITE brings together spike-in derived NGS data, annotations anddata evaluation in an easily navigable way.


PO 11Suppression before amplification of highlyabundant mRNA to profile gene expressionfrom microdissected mammary epithelial cells

Claudia Bevilacqua 1,∗, Nicolas Crapart 1,2,Cathy Hue-Beauvais 1, Barbara Brandao 1,Sophie Lemoine 3, Fanny Coulplier 3,Lucile Broseus 4, Patrice Martin 1

1 GABI, INRA, AgroParisTech, Université Paris-Saclay,Jouy-en-Josas, France2 EXCILONE, Elancourt 78990, France3 Plateforme de Génomique, Institut de Biologie del’ENS, IBENS, Paris F-75005, France4 INRA, UMR1213 Herbivores, F-63122 Saint-GenèsChampanelle, FranceE-mail address: [email protected]

Laser capture microdissection (LCM) is a powerful tool to isolatehomogenous cell populations from a heterogeneous tissue to ana-lyze their expression profile. Due to the small amount of materialcollected in such a way, an amplification step has to be carried out

dx.doi.org/10.1016/j.bdq.2017.02.097



dx.doi.org/10.1016/j.bdq.2017.02.098



dx.doi.org/10.1016/j.bdq.2017.02.099



S lar Det

bnolroqomstt2tawbt(Tavodltt(

h

PIqs

EJ

1

2

TE

oHtaeecgs

AFE4csr

pea

based on function, maturity, and other complex parameters suchas the timing of cytokine secretion.



efore library preparation, to obtain a specific gene expression sig-ature of microdissected cells using RNA-Seq. The main challengef any amplification protocol is to preserve the relative transcriptevels, enabling transcriptional levels and events to be faithfullyepresented. Today, several kits are available and each one is basedn different amplification strategies. The quality (RIN value) anduantity of RNA to start from play an important role in the choicef the amplification kit to use. Moreover, RNA-Seq approaches onammary epithelial cells (MEC) isolated by laser capture microdis-

ection (LCM) showed that, at the peak of lactation, ca. 3/4 of theranscripts correspond to the 6 genes encoding the 6 main milk pro-eins: 4 caseins, a-lactalbumin and b-lactoglobulin (Canovas et al.,014). The objectives of this study was to evaluate the possibilityo deplete the abundant transcripts prior amplification of cDNA forRNA-Seq analysis to access very low expressed genes. For thate used Ovation Human FFPE RNA-Seq Library Systems (Nugen)

ased on the Inda-C technology. The RNAseq results were comparedo that obtained with another kit based on the SMART technologySMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, Clontech).he objective was to evaluate the impact of depletion on sensitivitynd reproducibility starting from microdissected cells with a RINalue ranging between 6.5 and 7. Results show that the abundancef transcripts encoding the 6 major milk proteins in Nugen librariesepleted for both ribosomal RNA and milk protein transcripts was

owered to 3–4% of reads aligned on exons, compared to 75–80% inhe SMART-seq library. In contrast, SMART-seq kit allows to obtainranscriptomic profiles starting from really small amount of RNA1 ng) whereas 25 ng were necessary with the Ovation kit (Nugen).


O 12mplementation of a new automated sampleuality control tool in a whole exomeequencing workflow

lisa Viering 1, Jana Petersen 2, Marie Beckhaus 2,ana Molitor 1,∗, Stephan Wolf 2

Agilent Technologies, Inc., Waldbronn, GermanyDKFZ Genomics and Proteomics Core Facility, Highhroughput Sequencing Unit, Heidelberg, Germany-mail address: [email protected]

Objectives: The German Cancer Research Center (DKFZ) is onef the largest biomedical research institutions in Germany. Theigh Throughput Sequencing Unit of the DKFZ Genomics and Pro-

eomics Core Facility provides sequencing services for multiplepplications. This project demonstrates the use of an automatedlectrophoresis system as a quality control (QC) tool in a wholexome sequencing workflow. Mandatory for the experimental suc-ess of whole exome sequencing is the quality of the incomingenomic DNA (gDNA) material and the DNA samples at varioustages of the library preparation workflow.

Methodology: Exome libraries were prepared according to thegilent Low Input Sure-SelectXT Human All Exon v5 protocol fromFPE tumor tissue samples. The libraries were equimolar pooled.ach pool was sequenced on two lanes using the Illumina HiSeq000 System with 100 bp paired end sequencing. To ensure suc-ess quality control was verified with an Agilent 4200 TapeStationystem of the received gDNA samples and during the library prepa-ation.

Results: Intermediate QC steps were taken throughout therotocol to monitor library preparation for sequencing, such asvaluation of DNA after fragmentation, analysis of adapter-ligatednd amplified DNA, and lastly, qualification of the final library. The


initial QC of incoming gDNA was determined based on the DNAintegrity number (DIN). All samples had a low DNA integrity, whatis usual for DNA extracted from FPPE material. Due to the low qual-ity of the DNA material, a modified fragmentation protocol wasused. Modification of the fragmentation enabled to obtain mean-ingful sequencing results.

Conclusion: Quality control is an important part of NGS work-flows, library preparation protocols recommend quantification andqualification of the DNA samples at various stages. The increas-ing sample throughput creates a need for automation especiallyin a core facility where many precious samples are proceeded withtime pressure. The implementation of the automated electrophore-sis system in the whole exome sequencing workflow enabled toincrease the efficiency of the workflow and ensure good sequencingresults.


PO 13A SMARTer approach to profiling the humanT-cell receptor repertoire using the ICELL8single-cell system

Sara Gonzalez-Hilarion 1,∗, Sarah Taylor 2,Thomas Schaal 3, Jude Dunne 3,Maithreyan Srinivasan 3, Andrew Farmer 2

1 Takara Bio Europe, France2 Takara Bio, USA3 WaferGen Bio-Systems, Inc., USAE-mail address:sara [email protected]

Profiling T-cell receptor (TCR) diversity is critical for under-standing the adaptive immune system and can provide valu-able insights in studies involving immuno-oncology, immunedeficiency, autoimmunity, and vaccine response. While low-throughput approaches have yielded important insights concern-ing TCR repertoire dynamics, the development of next-generationsequencing (NGS) technologies has dramatically expanded theprospects for this research area. Using SMART

®technology, we

previously developed an NGS library-preparation method thatemploys a 5′ RACE-like approach to capture full-length variableregions of TCR-alpha and/or TCR-beta subunits. In contrast with TCRprofiling methods that use genomic DNA as starting material, ourSMARTer

®approach allows for analysis of expressed TCR sequences

that are likely to encode functional receptors and utilizes singleprimer pairs for each subunit, thereby avoiding amplification biasesassociated with multiplex PCR. This approach was originally devel-oped for RNA or bulk-cell inputs, requiring a minimum of 10 ngof RNA or 50 cells. In this poster, we present a modified librarypreparation protocol adapted for allowing high-throughput single-cell TCR-alpha/beta profiling using the WaferGen ICELL8 Single-CellSystem. This approach, in combination with phenotyping, enablesresearchers to identify specific pairings of TCR alpha and TCR betachains that comprise functional receptors in individual cells, andprovides a starting point for classifying these individual T-cells

dx.doi.org/10.1016/j.bdq.2017.02.100



dx.doi.org/10.1016/j.bdq.2017.02.101



dx.doi.org/10.1016/j.bdq.2017.02.102

lar Det

PWmhdpw

JOMTJTR

1

U2

CS3

E

cio(ctgflo

lhwagaS

bcgaO4hOlmo

aicpoprwiwf


O 14hen the promises and perils of advancedolecular diagnostics unexpectedly go hand in

and: Genomic analysis of uterine lavageetects early endometrial cancers and reveals arevalent cancer-driver mutation landscape inomen without cancer

ohn A. Martignetti 1,2,∗, Navya Nair 1,lga Camacho-Vanegas 1, Dmitry Rykunov 1,atthew Dashkoff 1, Sandra Camacho 1,

imothy Harkins 3, Cassie Schumacher 3,on Irish 3, Elena Pereira 1, Sviatoslav Kendall 1,amara Kalir 1, Nimesh Nagarsheth 1,obert Sebra 1, Boris Reva 1, Peter Dottino 1

Icahn School of Medicine at Mount Sinai, NY,nited StatesLaboratory of Translational Research, Westernonnecticut Health Network, Danbury, CT, UnitedtatesSwift Biosciences, Ann Arbor, MI, United States-mail address: [email protected]

Introduction: Endometrial cancer is the most common gyne-ologic malignancy and incidence and associated mortality arencreasing. Currently, there is no effective screening methodol-gy or protocol for endometrial cancer. The Cancer Genome AtlasTCGA) revealed many of the molecular defects that define thisancer. Based on these cancer genome results, and in a prospec-ive study, we hypothesized that the use of ultra-deep, targetedene sequencing could detect somatic mutations in uterine lavageuid obtained from women undergoing hysteroscopy as a meansf molecular screening and diagnosis.

Methods: Uterine lavage and paired blood samples were col-ected and analyzed from 102 consecutive patients undergoingysteroscopy and curettage for diagnostic evaluation. Lavage fluidas separated into cellular and acellular fractions and cellular

nd cell-free DNA were isolated, respectively. Two targeted next-eneration (NGS) gene panels, 56 genes and 12 genes, were usednd orthogonal mutation validation was performed using dPCR andanger sequencing.

Results: 7 patients were diagnosed with endometrial cancerased on classic histopathology following their hysteroscopy pro-edure. 6 had stage IA cancer. All 7 patients had cancer driverene mutations in lavage-isolated DNA and these mutations, abovespecific allele frequency, were present in the matched tumors.f the remaining 95 patients with benign/non-cancer pathology,3 had no significant cancer mutations. Intriguingly, 51 patientsad relatively high allele frequency, cancer-associated mutations.ne of these negative histopathology cases returned 6 months

ater with stage IA endometrial cancer. Increasing age and post-enopausal status were significantly associated with the presence

f driver/potential driver mutations.Conclusions: Our study demonstrates that an NGS-based

pproach of genomic analysis of lavage fluid has the sensitiv-ty to identify even early stage, microscopic foci of endometrialancer. Moreover, this genomic approach identified one canceratient, who originally had a negative histopathology diagnosisf tissue curettage, nearly 1 year prior to her returning with aositive histopathologic cancer diagnosis. Unexpectedly, we alsoeveal a prevalent landscape of cancer driver mutations in womenithout histopathologic evidence of endometrial cancer. Beyond

dentifying an opportunity to gain new insights into why someomen with cancer driver mutations remain healthy and cancer-

ree while others develop endometrial cancer, these studies provide


cautionary evidence of a Pandora’s box in NGS-based cancerdiagnostics.


PO 15Influence of genome update and methodologyon biomarker discovery – RNA-Seq study inchicken

Silvia Fibi 1,∗, Candida Vaz 2, Bertrand Grenier 1,Gerd Schatzmayr 1, Vivek Tanavde 2

1 Biomin Research Center, Biomin Holding GmbH,Technopark 1A, A-3430 Tulln, Austria2 Bioinformatics Institute, 30 Biopolis Street,Singapore 138671, SingaporeE-mail address: [email protected]

In October 2016, a new assembly of the chicken genome, Gal-gal5, was released with 9000 new annotated genes (coding andnon-coding). At that time, RNAseq data from a chicken study wasanalyzed with the former version Galgal4 to determine the mode ofaction of antibiotic in the intestine of birds, and evaluate potentialbiomarker(s). Therefore, it was necessary to reanalyze and comparethe outputs with both genome versions for discovery of potentialnew target genes. In addition, two alignment applications, STARAligner and TopHat, were assessed in our methodology.

Briefly, RNA was extracted from the ileum of chickens fed for35-days either basal feed (control) or supplemented with avil-amycin (antibiotic). Five biological samples per group (with twotechnical replicates per sample) were used for RNAseq (IlluminaHiSeq, stranded paired-end data, 30 M reads, 150 bp). Quality ofthe sequenced reads was evaluated in FastQC, and Trimmomaticwas used to remove adapters and filter the reads. TopHat andSTAR aligner were evaluated for alignments of the reads to thegenome. To quantify the genes, Cufflinks was used. The final out-puts were visualized and compared in Partek

®Genomics Suite to

obtain differentially expressed genes (DEG) (antibiotics vs. con-trol using a 1way-ANOVA model, p-value with FDR < 0.05, foldchange > 2 or <−2). The same pipelines were used to comparethe two genome versions (reads mapped with either Galgal4 orGalgal5).

The hierarchical clustering in Partek®

was the same for thefour final outputs (done with 2 aligners and 2 genomes). How-ever, two biological replicates in the control had to be excludedfor determining appropriately DEG between control and antibioticgroups. The total number of DEG per pipeline was as follows, Gal-gal4 in STAR = 139, in TopHat = 139, and Galgal5 in STAR = 142, inTopHat = 201. Different algorithms for the two aligners, and rean-notations in the genome Galgal5 could explain the discrepancy inthe results with TopHat.

When using STAR aligner, 55 DEG were found in commonbetween Galgal4 and Galgal5 (out of 139-142 DEG). Similarly, only47 DEG were commonly found between genome versions withTopHat (out of 139-201 DEG). When comparing STAR aligner andTopHat with one version of the genome, 111 DEG were commonlyfound with Galgal4, and 128 with Galgal5. The resulting number ofDEG in common across the four pipelines was 42. This shows thatboth aligners resulted in very similar outputs regarding DEG, butreads mapped with either Galgal4 or Galgal5 significantly affect thefinal results. A closer look revealed that many genes annotated in
Galgal4 disappeared from the entry system in ENSEMBL database.Corrections done in the new assembly of the genome and the rean-notation system greatly account for that. In conclusion, further


dx.doi.org/10.1016/j.bdq.2017.02.103



S lar Det

dp

h

PAs

E

E

atw(bDbii

idfto(ts

sasiaapantn

sismiietosa

h

chi, Gizem UYGUN, M. Türkan SAHIN who provided expertise that


ownstream analysis to discover potential biomarker(s) should beerformed with the results of Galgal5.


O 16voiding DNA adsorption to tube walls in NGSample preparation

mily Flowers, Hanna Oldfield ∗, Gerrit Gutzke

4titude Ltd., Germany-mail address: [email protected]

Polypropylene (PP) is the ideal plastic material for (q)PCR tubess PP is chemically inert, resistant to solvents and is well suitedo injection moulding, allowing for (q)PCR tubes to be producedith very thin walls for fast heat transfer and therefore optimum

q)PCR results. Despite the very hydrophobic nature of PP, DNA haseen shown to bind to PP tubes, especially in high ionic conditions.ifferent PP polymers can be used for producing PCR consuma-les, each of which have different characteristics. A particularly

mportant characteristic of PP polymers is the surface charge, whichnfluences binding of DNA in varying amounts.

Binding of DNA to PP is typically not an issue for (q)PCR as dur-ng amplification, any DNA stuck to the walls of the tube is releaseduring the denaturation step, maintaining accessibility of the DNAor the reaction. This is, however, becoming more of an issue due tohe progressing miniaturisation of reaction volumes and the devel-pment of new technologies such as Next Generation SequencingNGS). These techniques often use very small amounts of DNA andherefore require ultra-low DNA binding consumables to ensurepecificity and consistency of the reaction.

4titude®

Ltd. have developed both FrameStar®

PCR plates andtorage microplates that utilise a PP polymer with low binding char-cteristics which make them well suited for use in NGS and otherensitive techniques. No coatings are used to achieve the low bind-ng characteristics of the products ensuring samples are safe fromssay inhibition and degradation by leachables. Other options arevailable to reduce the DNA binding to tubes, including the use ofolyallomer or siliconised tubes; however, these are not as readilyvailable as PP and often demand a much higher price. Additionally,on-ionic detergents such as Triton-X and Tween 20 can be addedo samples at a low concentration to prevent DNA binding to PP butot impact on qPCR reactions.

On comparison with low binding products from other vendors,erial incubation of a 1.1 kb linear DNA fragment showed no signif-cant loss of DNA in 4titude low binding plates, while some loss waseen from low starting concentrations in plates made of alternativeaterials or competitor low binding branded plates. Additionally,

ncubation temperatures of 4 ◦C, 37 ◦C and 65 ◦C had no significantmpact on DNA binding. 4titude low binding plates offer ideal prop-rties for highly sensitive assays where low DNA input is vital andherefore any loss would have a significant impact on the successf the assay. Low binding plastics offer advantages not only for sen-
itive techniques such as NGS but also for the storage of samplesnd use in the forensic market.


PO 17An information system to support geneticallymodified organisms analysis strategies inTurkey

Remziye Yilmaz ∗, Ceren Cansın Satir

Hacettepe University, Food Engineering Department,06800 Beytepe, Ankara, TurkeyE-mail address: [email protected]

According to the laws in Turkey, food crop products (cereal, oilseed, fruits and vegetables, etc.) have to be analyzed as geneticallymodification (GMO) by law. There are many laboratories that needto have a specific strategy about GMO analysis. In this project, ouraim is to create Basic Information System for GMO Analysis to spec-ifying GMO analysis strategy with using DNA based methods. Withthe basic information system of GMO analysis, laboratory and allparties who requests for analysis can easily create their own GMOanalysis strategy. Basic information system for GMO analysis couldbe useful for laboratories and also provides optimization for riskassessment.

Molecular biology techniques used for fast, accurate, highlyrepeatable GMO content detection, identification and quantita-tion with multiple results are fundamentally divided into twogroups; DNA-based analytical techniques and protein-based ana-lytical techniques. The usage possibilities for traceability andcontrol of DNA, RNA and protein isolation methods, PCR and ELISAtechniques, DNA sequencing techniques, and finally microarraytechnique have been expanding increasingly. In terms of molecularbiology, food, feed and seed samples with multiple components, notonly plant or animal originated materials but also large and diffi-cult materials. It’s very important title of quick DNA, RNA or proteinisolation from every sample, with high quality and quantity. Nowa-days for GMO analysis; intensively used real time polymerase chainreaction technology (Real Time PCR, RT-PCR) have been acceptedat national and international level and essential to provide quanti-tative results to be obtained. At the national level, there are manylaboratories that need to have a specific strategy about GMO analy-sis. In this project, it’s aimed to create Basic Information System forGMO Analysis to specifying GMO analysis strategy. For this purpose,

Approved GMO types in Turkey, GMO types confirmed by Euro-pean Union GMO types and genes that can be analyzed in TurkeyStatuses, analysis contents and locations of related laboratoriesAt least one procedure for GMO analysis that has an internationalacceptance would be specified and placed on Information Systemof GMO Analysis. Under favor of Basic Information System ofGMO Analysis, laboratory and all parts who requests for analysiscan easily create their own GMO analysis technology. Usage ofFundamental Information System of National GMO Analysis wouldmakes procedure unity between laboratories and also providesoptimization for result assessment.

Acknowledgments: We are thankful to Hamideh Hammam-

greatly assisted in the research.


dx.doi.org/10.1016/j.bdq.2017.02.104



dx.doi.org/10.1016/j.bdq.2017.02.105



dx.doi.org/10.1016/j.bdq.2017.02.106

lar Det

PImt

BA

E

ism

hanTisWG

cwsGc

iu

F

F

F

wmgpakptta(i

Ctpwth

maDGa

h

not observed in 2016 as none of the 297 sampled plants was testedpositive for GM rapeseed.



O 18dentification and quantification of genetically

odified organisms (GMO) from highhroughput sequencing data

jørn Spilsberg ∗, Karin Lagesen,nja Bråthen Kristoffersen, Arne Holst-Jensen

Norwegian Veterinary Institute, Norway-mail address: [email protected]

We have explored the feasibility of detecting and quantify-ng genetically modified organisms (GMO) by high throughputequencing (HTS) analysis. The rapid drop in sequencing costs canake GMO analysis by HTS affordable and cost-effective.A GMO can be defined as an organism where the genetic material

as been altered in a way that does not occur naturally by matingnd/or natural recombination (EU DIRECTIVE 2001/18/EC). Thus,ot all GMOs contain gene(s) from another species (trans-gene).he GMO soybean line DP-305423 is genetically modified to alterts lipid metabolism via RNAi, and the inserted sequences are solelyoybean derived. This GMO can therefore be defined as a cis-gene.

e sequenced a 10% (mass/mass) sample of DP-305423 in a non-MO soybean background on an Illumina HiSeq.

A GMO is identifiable by the junctions between the artifi-ially inserted sequence and the flanking host genome. Each readas mapped to a database consisting of the inserts and flanking

equences of 43 GMOs plus the soybean genome (RefSeq accessionCF 000004515.4). Only unambiguous hits with MAPQ ≥ 20 wereonsidered.

We used expected frequencies to calculate the statistical signif-cance of reads bridging a junction. The following equations weresed to calculate the expected frequencies:

1 = 1 + (0.25)n×0.99 × (l − n) (1)

2 = (0.25)m×0.99 (2)

= F1 × F2 (3)

here n and m is the length of the part of the read that can beapped to one side of the junction and l is the length of the sample

enome (here = 1.115 Gbp for soybean; Eq. (1)). To place the firstart of the read, n, is equivalent the expected frequency of findingrandom n-mer anywhere in the soybean genome plus 1, since wenow that we have sequenced soybean and that the sequence isresent in soybean (Eq. (1)). When the first part is placed, is placinghe second part, m, equivalent to place a m-mer at a specific loca-ion in the soybean genome (Eq. (2)). Total expected frequency ofrandom read bridging the junction is the multiplied frequencies

Eq. (3)). We decided to apply a frequency threshold of ≤0.01 todentify significant reads and thus identify a GMO.

Coverage ratios can be used to estimate the GMO concentration.overage over the entire insert can be used for a trans-gene wherehe inserted sequence is absent in the host genome. This is notossible for a cis-gene where the inserted sequence is present else-here in the host genome. The junctions, however, are unique and

he coverage over the junctions relative to the coverage across theost genome can therefore be used to estimate the GMO content.

A total of 165,831,876 paired-end raw reads generated andapped to the GMO/soybean database with BBMap (version 36.59)

fter QC. Twenty-one reads were found to bridge the 8 junctions inP-305423 with a median expected frequency, F, of 1.8 × 10−39. The
MO content was estimated to 10.8%, which is close to the certifiedssigned concentration of 10%.


PO 19Optimized analysis procedure for monitoringtransgenic rapeseed along transportationroutes in Bavaria

Larissa Murr ∗, Patrick Guertler, Melanie Pavlovic,Ottmar Goerlich, Ulrich Busch

Bavarian Health and Food Safety Authority,Oberschleißheim, GermanyE-mail address: [email protected]

At present, no genetically modified (GM) rapeseed line (event)is authorized for commercial cultivation in the European Union,whereas some herbicide tolerant events are approved for their useas food and feed. Spillage of transgenic rapeseed during transporta-tion to processing plants would be considered as an unauthorizedrelease. The occurrence of transgenic rapeseed due to seed lossesduring transportation was already observed in several countrieswhere cultivation or even import of GM rapeseed is banned. There-fore, the presence of transgenic rapeseed lines along transportationroutes in Bavaria is being investigated within a project since 2014.For this purpose, rapeseed plants are sampled at sites where theprobability of seed spillage is comparatively high. In 2016, thesampling locations comprised an inland harbor in Bamberg, theaccess roads of the only central oil mill of Bavaria situated inStraubing as well as the vicinity of decentral oil mills. In the initialanalysis procedure, DNA isolation from rapeseed leaves was per-formed using an automated method, which was previously alignedto DNA extraction for leaves. Afterwards, an element-specific mul-tiplex qPCR assay was applied to limit the number of potentialevents which could be identified in subsequent event-specific sin-gleplex qPCR assays. Since this approach requires large amounts ofDNA and high expenditure of time, DNA extraction method fromleaf samples was optimized and five official event-specific single-plex qPCR assays were combined to a multiplex qPCR setup. Thepentaplex assay should allow the simultaneous detection of trans-genic rapeseed lines approved in the European Union for their useas food and feed: GT73, MON88302, MS8, RF3, MS8xRF3 and T45.Hydrolysis probes were labeled with different dyes (FAM, HEX,TEX615, Cy5, TAMRA). Fluorophores were chosen by considerationof their spectral properties. Overlapping of the emission spectrashould be kept as low as possible to avoid crosstalk between thedetection channels. Primer and probe concentrations were variedto determine optimized conditions for the detection of the five tar-gets. The multiplex qPCR assay was validated by examination ofthe limit of detection, the specificity, the robustness and the effi-ciency of the method. The limit of detection was five copies perreaction for each target. The individual detection systems fulfilledthe acceptance criteria for amplification efficiency and coefficientof determination. The multiplex qPCR assay showed high speci-ficity to the targeted sequences and was robust to small deliberatedeviations from the experimental conditions. The appliance of themethod for the analysis of the sampled rapeseed plants turned outto be time-effective and led to savings in DNA input. The occurrenceof transgenic rapeseed along transportation routes in Bavaria was



dx.doi.org/10.1016/j.bdq.2017.02.107



dx.doi.org/10.1016/j.bdq.2017.02.108

S lar Det

PDdJ

PH

1

O2

aE

faJI(

pvUcnBf

ubsowab

jgmEytdqaTos

ceNDAa

fAiv

h

ples prescribed by methodology, it is important to be familiar withnature of sample and its composition.



O 20evelopment of specific qPCR assays for theetection of genetically modified Alfalfa J101,

163 and KK179

atrick Guertler 1,∗, Melanie Pavlovic 1,eike Naumann 2, Ulrich Busch 1

Bavarian Health and Food Safety Authority,berschleissheim, GermanyLower Saxony State Office for Consumer Protectionnd Food Safety, Braunschweig, Germany-mail address: [email protected]

Alfalfa (Medicago sativa), as one of the world’s most importantorage crops, was genetically modified (gm) in order to gain toler-nce against the herbicide glyphosate (Round Up; events J101 and163) or to alter the lignin composition of the plant (event KK-179).n 2015, about 13% of the total alfalfa cultivation in the United StatesUS) was gm.

A recent study has shown the spreading of gm alfalfa on ferallant sites in the US, which might lead to a contamination of con-entional alfalfa products, which are also exported to the Europeannion (EU). Biotech crops need to be authorized for import orultivation in the EU and according to the non-tolerance policy,on-authorized gm crops may not be placed on the EU market.iotech alfalfa is not deregulated in the EU and is therefore in the

ocus of official surveillance authorities.Detection and identification of gm crops is currently performed

sing real-time PCR (qPCR) assays, targeting the junction sequencesetween the plant genome and the inserted DNA fragment (event-pecific detection). Up to now, no qPCR assays for the detectionf gm alfalfa and no reference material are available. Therefore,e designed plasmids as reference material and developed qPCR

ssays for the detection and identification of three alfalfa eventsased on patent sequence information.

The plasmids for events J101 and J163 contain the 5′ and 3′

unction sequence, a Col E1 origin and a kanamycin resistanceene. The KK179 plasmid contains the whole inserted DNA frag-ent and the 5′ and 3′ junction sequences, as well as a Col

1 origin and an ampicillin resistance gene. Primers and hydrol-sis probes were designed targeting the junction sequences ofhe respective alfalfa events. The assays were in-house vali-ated using a 7900HT instrument and the Promega GoTaq ProbePCR Master Mix. Probes were labelled with a FAM reporter dyend two quenchers (ZEN: internal quencher; IBFQ: 3′ quencher).he following assay specifications were determined: optimalligo concentration, limit of detection, robustness, efficiency andpecificity.

Robustness tests indicate very stable assay performance afterhanging the master mix and after varying performance param-ters (master mix volume ±1 �L, annealing temperature ±1 ◦C).o unspecific amplification signal was observed after analysis ofNA from different plant species and from other biotech crops.nalyses revealed a limit of detection of 5 copies/PCR for allssays.

A ring trial still needs to be set up in order to show the trans-erability of the assays and to verify the optimized PCR conditions.s soon as certified reference material for the three alfalfa events

s available, the developed detection systems should be applied toerify their suitability with actual sample material.



PO 21GMO analysis of soy lecithin and productscontaining soy lecithin

Danica Milinkov Guljas, Jelena Hrubik ∗,Gordana Novic

SP Laboratorija, SerbiaE-mail address:[email protected]

Lecithin is a natural lipid that can be found in all living cellsand it is primarily obtained from plants that contain oils, primarilysoybean. It is used for applications in food mostly as emulsifier,feed and pharmaceuticals. The raw lecithin is separated from thesoybean oil, and further can be purified in several steps.

In practice, lecithin counts as a difficult matrix for DNA isola-tion. Using of the CTAB based DNA isolation protocol for lecithin inusual way, as it is applied for other samples routinely, will resultin poor quality and concentration of DNA which would be inappli-cable for real time PCR reaction and further genetic modifications(GM) analyses. The quality of DNA depends on the processing levelto which raw lecithin was subjected.

Adjusting the isolation procedure to this type of samplesbrought positive results, and it was examined and confirmed bythe several dozens of samples. The most successful results wereobtained when the whole amount of the supernatant in first stepsof isolation was used, while merely changes in sample weighs didnot give the desired results. The DNA concentration isolated in suchway was >10 ng/�l, and the purity was in acceptable range. The sat-isfying DNA isolate was confirmed with lectin (soy taxon specificgene) amplification curves detected in the real time PCR reaction.

Difficulties emerge in analyses of samples containing smallamounts of lecithin, such as products supplemented with soylecithin, as well as products that contain only a several percent oflecithin such as chocolates, cookies, wafers etc. Isolation of DNAfrom cookies containing soy lecithin as emulsifier, resulted in agood quality and quantity of DNA (>30 ng/�l), but lectins were notamplified in real time PCR reaction.

This problem has been overcome by product partitioning in theeasily separable units which contain lecithin. Further, the isola-tion procedure was adjusted in the same manner as it was donefor lecithin. From these separate product units, the sufficient quan-tity and quality of soy DNA was obtained, which was confirmed byamplification of lectins in real time PCR reaction.

Investigation and confirmation of above statements were per-formed on 42 samples of different products during 2016. Allexamined products contain less than 5% lecithin, as the only soy-derived component in them. In 40 cases (95%) it was possible tocomplete the GM analyses.

In conclusion, the expertise of analysts and a good knowledge ofthe principles and techniques of GM testing methods, enable adap-tation of DNA isolation procedure to specificities of sample. Also,in the analyses of GM, in addition to the basic analytical princi-



dx.doi.org/10.1016/j.bdq.2017.02.109



dx.doi.org/10.1016/j.bdq.2017.02.110

lar Det

PBe

L

E

ci(cil

tcPds(

cDlce

ec

h

PAfd

AS

E

gtp(tirpttdtrtpoCcs

automation solution delivers reproducible automated pipetting,while reducing record keeping errors and user-to-user variability.



O 22iocev GeneCore: More than just genexpression profiling

ucie Langerova ∗, David Svec

Institute of Biotechnology, Czech Republic-mail address: [email protected]

We present services of BIOCEV Gene Core – the best equippedore facility and service provider in the field of gene expressionn Central Europe. We have broad experience in quality controlQC, e.g. Fragment Analyser) in a single cell analysis (automatedell picking ALS Cellcelector), high-throughput and digital PCR (Flu-digm Biomark, BioRad QX200 Droplet Digital PCR System) and NGSibrary preparation.

We emphasise quality control, which is often neglected. Effec-ive QC is based on the use of molecular tools to controlontamination (RNA/DNA spikes), genomic background (Valid-rime) and quality of RNA (�Amp, RIN). We also take part inevelopment of these methods to facilitate analysis of gene expres-ion starting from bulk samples, down to the level of individual cellsdirect lysis).

In addition to conventional qPCR analysis, we focus on singleells expression profiling and multi-analyte approach. Analysis ofNA/RNA/protein in parallel in one sample even on the single cell

evel provides comprehensive tool to map gene expression andharacterizes types of cells and to determine the degree of differ-ntiation and to study the pathological condition.

Currently we offer assistance with library preparations andxperimental design of RNA-Seq experiments, which are key pre-onditions for a successful project.


O 23utomated, high-throughput quantitative PCR

or accurate and economical copy numberetermination

lexander Kolb, Luke Linz, Ben Sowers,tephan Bauer ∗

LGC, United States-mail address: [email protected]

Copy number variation (CNV) has become a popular area ofenetic research with an increasing number of studies highlightinghe importance of CNV in relation to human health. One exam-le in the field of pharmacogenomics is the cytochrome P450 2D6CYP2D6) gene. Deletions and duplications of CYP2D6 can impacthe metabolism of many clinically relevant drugs, therefore mak-ng it a popular target for continued research. The IntelliQube

®

eal-time qPCR instrument in conjunction with dual labeled BHQ®

robe-based assays provides an accurate, high-throughput solu-ion for assessing CYPD26 CNV. Utilizing Array Tape

®technology,

he IntelliQube integrates liquid dispensing, thermal cycling, andetection with qPCR analysis in miniaturized reaction volumes. Inhis study, we assess the performance of custom BHQ qPCR assaysun on the IntelliQube for determination of multiple copy numberargets within the CYP2D6 gene. Accuracy and reproducibility of thelatform was assessed using purified gDNA samples from cell linesbtained from the NIGMS Human Genetic Cell Repository at the
oriell Institute for Medical Research. Copy number results wereompared to previously published literature. The results demon-trate that the IntelliQube, when used in conjunction with BHQ

probes, provides a powerful solution for accurate, automated, high-throughput CNV research and analysis.


PO 24Comparison of manual vs. automated qPCRreaction plate setup

Tristan J. Berto ∗, Amber Mael, Laura Simdon,Michael Herren

Gilson Inc., United StatesE-mail address: [email protected]

Quantitative real time polymerase chain reaction (qPCR) is apowerful technique for detecting differences in the copy number ofnucleic acids in biological samples. qPCR techniques may be pairedwith reverse transcription and are used for applications such astranscript expression, detection of viruses, copy number variation(CNV), food safety testing and more. Each step in the extremelysensitive qPCR procedure can potentially contribute to experimen-tal error. Accurate and precise pipetting can minimize sources ofvariability. In this work we compared qPCR results for reactionplates set up using manual pipetting or automated using GilsonPIPETMAX

®qPCR Assistant v1.3. PIPETMAX is a robotic pipetting

workstation with motorized multichannel PIPETMAN®

technologyaccommodating volumes from 1 to 200 �L. The system supportsmany types of labware including as microfuge tubes, reservoirs,strip tubes, 96 well plates and 384 well plates. The software inter-face, TRILUTION

®micro v2.3, runs on a tablet or PC and includes

wizard-style assistants that automate common laboratory proce-dures. PIPETMAX qPCR Assistant automates plate preparation forqPCR, including preparation of master mixes, standard curve andsample dilutions, and reaction setup. The qPCR Assistant interfaceguides the user through experimental parameters including selec-tion of controls, dilution factors, and number of replicates, andprimer/template combinations. qPCR Assistant generates run doc-umentation that can be incorporated into laboratory notebooks,including master mix recipes, a detailed pipetting guide, list oflabware used in the experiment, and graphical representations ofreaction plate configuration. Additionally, the software generatesthermocycler run files that are compatible with all major real-timePCR instruments; this permits direct import of the sample informa-tion into the thermocycler and eliminates the need for data entryat that step, resulting in significant time savings for the user whileassuring traceability of the data. qPCR reactions were prepared in384 well plates (manually or using qPCR Assistant) to assess tran-script levels in a universal cDNA as well as human cDNA samplesfrom seven tissue types. Primer pairs were chosen from a panel ofpretested assays for human reference genes. Transcript levels, asassessed by Cq value, were comparable for reactions set up usingmanual or automated pipetting. Variance was lower for reactionsprepared on PIPETMAX. PIPETMAX qPCR Assistant fits into the qPCRworkflow and provide standardized and reliable methods to helplabs meet the MIQE publication requirements. This easy to use



dx.doi.org/10.1016/j.bdq.2017.02.111



dx.doi.org/10.1016/j.bdq.2017.02.112



dx.doi.org/10.1016/j.bdq.2017.02.113

S lar Det

POfq

RKRG

1

EM12

H3

RK4

MU5

SL6

E7

HK8

DME

iTntifioiiNaltsoctsttaPfudmodtma

sion and contraction as the VST of this material is 147 ◦C and thecoefficient of thermal expansion is 70 × 10−6 K−1.


O 25pening Pandora’s box – Unmasking the need

or a continuous sample processing control forPCR based fecal marker analysis

ita Linke 1,∗, Sibel Zeki 1,4, René Mayer 1,2,atharina Keiblinger 5, Simone Ixenmaier 1,2,obert Mach 6, Regina Sommer 2,7,eorg Reischer 1,8, Andreas Farnleitner 1,2,3

TU Wien, Institute of Chemical and Biologicalngineering, Research Group Environmentalicrobiology and Molecular Diagnostics 166/5/4,

060 Vienna, AustriaInteruniversity Cooperation Centre for Water andealth: www.waterandhealth.at, AustriaKarl Landsteiner University of Health Sciences,esearch Unit Water Quality and Health, 3500rems, AustriaDepartment of Marine Environment, Institute ofarine Sciences and Management, Istanbulniversity, Istanbul, TurkeyInstitute of Soil Research, Department of Forest andoil Sciences, University of Natural Resources andife Sciences, Vienna, AustriaTU Wien, Institute of Chemical and Biologicalngineering, 1060 Vienna, AustriaMedical University Vienna, Clinical Institute ofygiene and Medical Microbiology, Water Hygiene,inderspitalgasse 15, Vienna, AustriaIFA Tulln, Center for Analytical Chemistry,epartment of Agrobiotechnology, Working Groupolecular Diagnostics, 3430 Tulln, Austria


PCR based techniques for the rapid detection of trace level DNAn environmental samples have gained considerable popularity.he method of qPCR itself has become a widely used routine tech-ology and the challenges now centre on numerous limitations ofhe pre- and post-assay workflows. The aim of this study was themplementation of a detailed process control for the bacterial cellltration, DNA-extraction and qPCR-performance characteristicsf water samples from a habitat with a very diverse and chang-ng matrix composition to provide a robust basis for confirmednterpretation of qPCR data. The aquatic habitat selected was Lakeeusiedl, located in the Eastern part of Austria, an important recre-tion area for the large urban area of Vienna. High and fluctuatingevels of salt, humic acid, and suspended solids (TSS) characterizehe water matrix of Lake Neusiedl. Therefore, this habitat repre-ents a perfect model to study the basic performance characteristicsf qPCR-based diagnostics on water samples with changing matrixonditions. The recovery efficiency through the processes of extrac-ion and purification has only rarely been monitored for wholeample sets and when recovery efficiencies have been recordedhey ranged from 1 to 100%. In our study we therefore added a fil-ration/extraction process control to each sample by spiking withdefined cell standard (DeTaCS). In addition, a strict evaluation ofCR-inhibition by using an internal amplification control and dif-erent dilutions for each sample was performed. DNA was extractedsing a phenol-chloroform and CTAB based method. Results clearlyemonstrated that inhibition of the PCR reaction played only ainor role in the samples investigated. In contrast, the presence

f high amounts of (inorganic) TSS led to a complete loss of DNA
uring the process of DNA-extraction (i.e. recovery rates ≤1%). Toest the hypothesis that the adsorption of DNA to surface-reactive
atrix components during DNA-extraction was the causing mech-nism, spiking experiments with different amounts of sodium


pyrophosphate and salmon sperm were conducted in the labo-ratory. Addition of these adsorption site competitors helped torecover DNA concentrations to near control levels, however, withdifferent effects on qPCR performance. Higher amounts of sodiumpyrophosphate resulted in an inhibition of the PCR reaction. Theresults of the presented study impressively show the importanceof using a stringent and continuous process control on a “sample-per-sample” basis. Using this approach we were able to unmaskthe challenges of diverse matrices of samples from a single habi-tat and could adapt the DNA-extraction protocol in order to getcomparable qPCR results. We postulate that the herein observedchallenges are rather the rule than the exception and thus pro-pose the continuous use of a stringent filtration/extraction processcontrol as basis for a robust quality management of qPCR-basedenvironmental diagnostics.


PO 26Influence of PCR consumables on the accuracyof real-time PCR experiments

Gerrit Gutzke, Hanna Oldfield ∗, Emily Flowers

4titude Ltd, GermanyE-mail address: [email protected]

Classical PCR and qPCR plates are one-component plates madeout of polypropylene (PP). PP is the best plastic material for PCRtubes as it is chemically inert and allows for the production of ultra-thin tube walls which is important for fast temperature transfer.While PP has become the standard material for PCR consumablessome of its properties question its suitability for applications likeqPCR and NGS.

The material characteristics of PP exhibit a Vicat Softening Tem-perature (VST) of 90 ◦C and a coefficient of thermal expansion of180 × 10−6 K−1 which are potential weaknesses for its usage attypical (q)PCR temperatures.

When used for (q)PCR, not only do the plates soften during thedenaturation step, but measurements also show that the platesexpand by up to 2 mm in the diagonal plane (from room tempera-ture to 95 ◦C) and they shrink again as the temperature decreases.Therefore, the plate will undergo expansion and contraction inevery cycle, placing significant tension on the plate seals. As a result,contact between the seal and plate will be particularly weakenedin the corner positions and outer rows leading to evaporation fromthe plate in these areas, while centre wells will only be affectedminimally. This differential evaporation effect is especially eminentwhen adhesive seals are used (as opposed to heat seals).

Evaporation has a significant effect on the reaction conditionsresulting in noticeable effects, especially for qPCR. Identical sam-ples can exhibit significant differences in their Ct values, dependingon their position on the plate. This often remains unnoticed as trip-licates are typically placed in neighbouring wells which are affectedby similar levels of evaporation.

A solution to the problem of evaporation related qPCR inaccu-racies is the usage of two-component plates. These plates consistof tubes made out of PP but a frame made out of polycarbonate(PC). PC does not show significant temperature-dependant expan-




dx.doi.org/10.1016/j.bdq.2017.02.114



dx.doi.org/10.1016/j.bdq.2017.02.115

lar Det

PCOe

JF

E

ciaalsoo

aPbopa

fgbbfr(iE(

yRcRab

h

PCp

AF

1

C2

tCE

ambt

[

[

[

[


O 27ell-free DNA assessment for clinical purposes:ptimization of blood collection and DNAxtraction methodologies

os Drabbels ∗, Berit Kemps, Marijke Spruyt,rans Claas, Michael Eikmans

Leiden University Medical Center, The Netherlands-mail address: [email protected]

Background: Assessment and characterization of circulating,ell-free (cf)DNA is a promising tool in various clinical settings,ncluding prenatal diagnosis, detection of metastasizing tumors,nd detection of rejection after organ transplantation. The reli-bility of this tool depends on the specificity by which theow-frequency genotype (either fetal-, tumor- or donor-organ-pecific) can be quantified within the total circulating DNA poolf the host. Reliable assessment of cfDNA content first requiresptimization of the protocol.

Methods: We collected blood of healthy volunteers in differentnti-coagulant tubes (Heparin, Citrate, regular EDTA, EDTA-Streck,AXgene), and incubated for several time periods (0 h, 24 h, 48 h)efore cfDNA extraction using the genomic (g)DNA blood mini kitr circulating nucleic acid (CNA) kit (both from Qiagen) from thelasma. Total DNA yields were determined by a Qubit fluorometer,nd HCK gene copies were quantified by digital droplet PCR.

Results: The use of the CNA kit resulted in an average 2.1-old higher cfDNA yield (∼5.5 ng/mL of plasma) compared to theDNA kit. The type of blood tubes used and prolonged blood incu-ation may cause release of gDNA from the patient’s nucleatedlood cells, thereby possibly affecting specificity in detecting lowrequency targets. With Streck and PAXgene tubes, DNA yieldsemained constant between 0 and 48 hours blood incubation1.2 ± 0.4 fold). The use of Heparin tubes resulted in the highestncrease (24 h: 16.4 ± 19.0 fold; 48 h: 46.2 ± 70.8 fold), whereasDTA tubes resulted only in a slight fold increase after incubation24 h: 1.7 ± 0.1 fold; 48 h: 3.4 ± 0.5 fold).

Conclusion: The specialized CNA kit gives enhanced cfDNAields and is preferred for downstream processing of blood plasma.esults for Streck and PAXgene confirm that these preservant-ontaining tubes prevent gDNA release from white blood cells.egular EDTA tubes may offer a cost-effective alternative for cfDNAnalysis of blood plasma, processed within 24 h, which already haseen archived in laboratories.


O 28ompatibility of magnetic nanoparticles witholymerase chain reaction

yse Beyza Aysan 1,∗, Zdenek Knejzlík 1,2,rantisek Stepánek 1

University of Chemistry and Technology, Prague,zech RepublicInstitute of Organic Chemistry and Biochemistry of

he Czech Academy of Sciences of the Czech Republic,zech Republic-mail address: [email protected]

Nanoscale materials have seen a rapid increase in interest for the
rea of biotechnology and biomedicine. An important class of nano-aterials is nanoparticles based on magnetic materials, which can
e manipulated by an external alternating magnetic field. Owingo their exceptional properties they serve promising biomedical


application such as a contrast agent for magnetic resonance imag-ing (MRI), drug/gene carriers for targeted drug/gene, therapeuticagents for hyperthermia, or tissue repair. The modelling and themimicking of biochemical processes that combine nanoparticles isa new and interesting area of research [1–3].

The aim of this work is to design and implement microscopicbiochemical reactors, in the form of cell-sized capsules that canperform biochemical reactions such as the reproduction and ampli-fication of nucleic acids in vitro through the polymerase chainreaction (PCR) and be controllable remotely in a contactless way.Magnetic iron oxide nanoparticles (MNPs) which act as the localsource of heating by exposing to an RF alternating magnetic field arecombined with the PCR mixture (DNA template, enzyme, primers,dNTPs, etc.) within core-shell capsules. In this specific contribution,we present an investigation of the compatibility of the MNPs withthe PCR reaction components and identify the relevant conditionsfor effective PCR even in the presence of high concentrations ofMNPs.

MNPs were fabricated successfully [4] with various typesof functional groups including sodium citrate dehydrate(Na3C6H5O7·2H2O), dextran 70 (from Leuconostoc spp., approx.Mw 70 kDa), 3-aminopropyl-triethoxysilane (APTES) and char-acterized in terms of concentration, size distribution, surfacemorphology, and radiofrequency heating ability. The responseto the RF alternating magnetic field is proportional to the sizeand concentration of the nanoparticles. Results have shown thatdextran coated MNPs were stable in the PCR reaction mixtureeven in high concentration while the remaining types formedaggregates. Future experiments are focused on dextran coatedMNPs in sufficiently high concentrations to achieve the requiredPCR temperature by exposure to an RF alternating magnetic field.References

1] M. Mahmoudi, S. Sant, B. Wang, S. Laurent, T. Sen, Superparamagnetic ironoxide nanoparticles (SPIONs): development, surface modification andapplications in chemotherapy, Adv. Drug Deliv. Rev. 63 (2011) 24–46.

2] O.C. Farokhzad, R. Langer, Impact of nanotechnology on drug delivery, ACSNano 3 (1) (2009) 16–20.

3] A.K. Gupta, M. Gupta, Synthesis and surface engineering of iron oxidenanoparticles for biomedical applications, Biomaterials 26 (2005) 3995–4021.

4] A.B. Aysan, Z. Knejzlík, P. Ulbrich, M. Soltys, A. Zadrazil, F. Stepánek, Effect ofsurface functionalisation on the interaction of iron oxide nanoparticles withpolymerase chain reaction, Colloids Surf. B: Biointerfaces 153 (2017) 69–76.


PO 29Conception and optimization of a detectionmethod of Filovirus species by combining theuse of inosines and HRM analysis

Graziella Penot, Laure Barbier, Charles Chapus,Thomas Poyot ∗

IRBA, FranceE-mail address: [email protected]

Our institute develops dual research to respond both to thearmed forces and to the civilian uses. Different research programsare thus conducted in the field of Nuclear Biological Chemical (NBC)risks. Our unit is a core facility that offers various molecular biol-ogy tools, with the associated technical expertise. We also developinnovative research programs, linked to the NBC fields. The aim ofthis study was to develop an original method to quickly detect andidentify viral species, in a small biological sample. More precisely,
we focused on the detection of a Filovirus species, in a case of asuspect viral haemorrhagic fever. We have tried to design a panFilovirus PCR primer pair which can detect all selected Filovirusspecies, by using “universal base”, inosine or 5-nitroindole and


dx.doi.org/10.1016/j.bdq.2017.02.116



dx.doi.org/10.1016/j.bdq.2017.02.117



S lar Det

td

aTehhuab

IntpoaIgtr

icfnr

at

h

PEfor

SAS

1

ES2

CB3

PWE

maecmp

rnd


he High Resolution Melting curves analysis. The different primersesigned were then tested on a synthetic sequence.

The first step was to find the best zones on 5 Ebola genus speciesnd 4 Marburg genus species to design these pan Filovirus primers.he Filoviridae family presents a 19 kb genome, in the form of a lin-ar single stranded RNA, that codes for seven genes. If the functionalomology is strong, the nucleotide homology is quite low. Only 2omology zones were found to be compatible with the design ofniversal primers. We have designed two pairs of primers (for anverage size of 20 nucleotides), where 5 to 8 bases were replacedy a “universal base”: inosine or 5-nitroindole.

The second step was to test and validate these primers pairs.t has soon appeared that the primers containing 5-nitroindole didot allow any amplification. Several steps of optimization and adap-ation were necessary to obtain a valid and reliable amplification forrimers containing inosines. We began to test these optimizationsn one species: ZEBOV, in order to get reliable amplification. Oncen optimized condition was defined, we applied it to all the species.t appeared that the primer pairs containing less inosine basesave better amplification than the other. This condition allowedo amplify all species, even if the Tm profiles were not perfectlyeproducible for all of them.

In order to improve the reproducibility and the sensibil-ty, another strategy was chosen: testing a semi nested PCR. Itonsists in a first PCR of 35 cycles to preamplify all matrices,ollowed by a second PCR on these diluted products. With thisew strategy, we obtained an increased sensibility and a bettereproducibility.

In conclusion, the use of inosine in PCR primers allows to amplifyll selected Filovirus species. Further investigations are necessaryo carry out this detection strategy.


O 30valuation of SNPs based on DNA extractedrom urine as part of the development and usef a kit of non-invasive biomarkers to monitorespiratory health of young children

arah Nauwelaerts 1,2,∗, Koen De Cremer 2,lfred Bernard 3, Nancy Roosens 1,igrid De Keersmaecker 1

Platform Biotechnology and Molecular Biology DOxpertise, Service Provision and Customer Relations,cientific Institute of Public Health, Brussels, BelgiumHealth and Environment DO Food, Medicines andonsumer Safety, Scientific Institute of Public Health,russels, BelgiumLouvain Centre for Toxicology and Appliedharmacology, Université Catholique Louvainoluwe, Brussels, Belgium


Respiratory health of children is among the priorities of environ-ental health programs. Both genetic and environmental factors

re affecting the children’s likelihood to develop respiratory dis-ases. Monitoring exposure, effect and susceptibility in children’sohorts, using biomarkers is of critical importance for health careanagement purposes, public health decision making, and primary

revention activities.
The most commonly used source of biomarkers is serum but it
equires blood sampling, which for ethical and practical reasons isot feasible in most epidemiological studies involving young chil-ren. Therefore, using non-invasive biomarkers like nasal lavage


fluid (NALF) or urine is a necessary alternative for monitoring theirrespiratory health. NALF reflects upper airways alterations foundin nasal epithelium while changes occurring in the deep lung areonly accessible through the urine as a source of protein biomarkers.Some studies have associated genetic polymorphisms to the devel-opment of hypersensitivity response or sickness. Furthermore, fewprospective studies have investigated how epigenetics affected byenvironmental exposure influences the health response. Futurebiomonitoring studies should integrate all proteinaceous, geneticand epigenetic aspects to optimize the use of health biomarkers andconsequently to reduce the potential risk of respiratory disordersfor substrata of the population.

Our study involves an existing biobank of 850 non-invasivesamples (NALF & urine) coming from 5-years old children linkedwith a questionnaire including data of environmental and lifestylefactors and respiratory health data. Currently, specific genetic mod-ifications correlated with the development of asthma are beingmeasured in the children’s biobank. Following the DNA extractionfrom urine, real-time PCR, is being used for the detection of DNApolymorphisms (e.g. SNP CC16 A38G). Statistical analysis will beperformed to correlate frequencies of the SNPs with risks of aller-gic sensitization of children. In a second stage, protein biomarkers(e.g. CC16) related to respiratory health will be measured with theMRM-technology. Additionally, epigenetic modifications poten-tially related to adaptive response to environmental insults or torespiratory health will be explored. These will include gene methyl-ation and the expression of miRNAs. Exosome isolation from NALFand urine will be explored in order to “concentrate” potentiallyunquantifiable or undetectable biomarkers.

The final goal of this project is to design a kit of non-invasive pro-tein, genetic and epigenetic biomarkers for allowing a better surveyand diagnosis of the respiratory health of children at an early stage.This information is necessary to support a pro-active public healthpolicy with the aim of improving respiratory health of children,and hence of older people with emerging health disorders whichare suspected to have their origin in young children.


PO 31Modaplex, a novel technology for the multiplexanalyses of CpG and miRNA signatures in asingle qPCR run

Diana Alejandra Grohme 1,∗, Kerstin Korn 1,Juliane Rothe 2, Ruth Merkle 3, Rongxi Yang 3

1 Biotype Innovation GmbH, Moritzburger Weg 67,01109 Dresden, Germany2 Biotype Diagnostic GmbH, Moritzburger Weg 67,01109 Dresden, Germany3 Department of Gynecology and Obstetrics,University Women’s Hospital of Heidelberg, 69120Heidelberg, GermanyE-mail address:[email protected]

Background: As the hallmark of cancer, DNA methylation isknown to be widely involved in the processing of cancer. microR-NAs are a group of small RNAs which can moderate the expressionof mRNAs, and thus involved in cancer related pathways. Although,there are currently several techniques available to analyse DNA
methylation and miRNAs, most of them are based on singleplexmeasurements. The development of biomarkers using multipleDNA methylation and miRNA signatures calls for a reliable mul-tiplex method that can analyse several DNA methylation sites and
dx.doi.org/10.1016/j.bdq.2017.02.118



dx.doi.org/10.1016/j.bdq.2017.02.119



lar Det

mnaCn

icmwwr

rmdvTwthsb

assyhr

o

h

PMs

OM

1

DB2

TE

pmrefp

PwawtIt4w


iRNAs in a single platform. Here, we present the Modaplex tech-ique, a novel and sensitive multiplex technology, which allows forsimple and rapid interrogation of the methylation status of fourpG sites and the relative quantification of four miRNAs simulta-eously in a single PCR run.

Methods: For relative quantification (1) a CpG multiplex assayncluding one control for highly methylated status and one bisulfiteonversion control was designed to amplify methylated and non-ethylated genomic DNA, and (2) an assay for amplifying miRNA asell as two reference miRNAs and one miRNA degradation controlas developed. The universal run protocol allows both assays to be

un simultaneously using the QIAGEN Modaplex Technology.Results: The Modaplex Methylation Assay* was designed for

elative quantification of the amount of methylated vs. non-ethylated DNA for four relevant CpG sites. Assay capability to

etect the methylation status was determined using bisulfite con-erted methylated and non-methylated DNA from human blood.he Modaplex miRNA Assay* is also a relative quantification assay,hich determines the expression of four miRNA markers. In order

o test the capability of the miRNA assay, miRNAs from cell lines anduman plasma/blood were used. First performance characteristictudies involving amplification efficiency, and limit of detection ofoth assays were carried out on human sample material.

Conclusion: The discovery of an altered epigenetic status suchs DNA methylation and miRNA expression during carcinogene-is might provide new markers for diagnosis. Development of aingle platform that can simultaneously analyse multiple meth-lation sites and miRNA expressions with a fast turnaround timeas the potential to facilitate diagnostical and therapeutical clinicalesearch.

* Modaplex methylation and miRNA assays are for research usenly. Not for clinical diagnostic use.


O 32olecular diagnosis of toxoplasmosis in

erologically positive pregnant women

uassila Aouacheri 1,∗, Hajira Berredjem 1,eryem Benlaifa 1, Rafika Bardi 2

Applied Biochemistry and Microbiology Laboratory,epartment of Biochemistry, Faculty of Sciences,adji Mokhtar University, Annaba, AlgeriaLaboratory of Immunology, EPS Charles Nicolle,unis, Tunisia-mail address: [email protected]

Recent acquired T. gondii infection is a serious problem inregnant women which present a high risk of Toxoplasma trans-ission to the fetus with serious damages. Serological diagnosis

elies on the detection of anti-Toxoplasma immunoglobulin; how-ver, serology may be unreliable especially when serological testingor IgG/IgM antibodies fail to differentiate between a recent and aast infection. In this case, we rely on PCR technique.

The purpose of this study was to compare serology assay andCR using B1 gene for the early detection of T. gondii in pregnantomen. Sera samples were collected from 85 pregnant women

nd measured by ELISA for specific IgG and IgM antibodies. DNAas extracted from 17 PBL samples and then amplified by PCR. A

otal of 41 out of 85 women was seropositive: 24 (58.53%) were
gG+/IgM− and 17 (41.46%) were IgG+/IgM+. PCR assay contributeo confirm toxoplasmic infection and detected Toxoplasma DNA inwomen presenting IgG+/IgM+. All samples from control patientsere negative. This study demonstrates the usefulness of the PCR

in the diagnosis of toxoplasmosis pregnant women. This methodmay be suitable for routine screening of T. gondii infection in theclinical laboratory in association with serological tests.


PO 33qRT-PCR methods to evaluate the seasonalvariations of relative expression of testicularLhcgR mRNA in adult male Libyan Jird(Meriones libycus)

Radia Boufermes

BADJI Mokhtar University of Annaba, AlgeriaE-mail address: [email protected]

The real time quantitative PCR was used to investigate the sea-sonal variations of testicular LhcgR mRNA expression in desertrodent (Meriones libycus) during the breeding and non-breedingseasons. We used the relative SYBR Green compared to endogenousgene beta actin expression method. This technique was used for thefirst time to evaluate gene expression level in this species. The rela-tive Lhcgr mRNA expression level was higher during non-breedingseason (2.10 × 10−3 ± 0.35 × 10−3) than during the breeding season(0.92 × 10−3 ± 0.15 × 10−3) (p < 0.05). Q.RT-PCR gave us the sensi-tive rate of gene expression to study the seasonal variations ofLhcgR expression in Libyan Jird.


PO 34Real-time PCR to evaluate the effect of theN-acylsulfonamide bis-oxazolidin-2-ones onToxoplasma gondii’s infected Swiss mice

Hajira Berredjem 1,∗, Meryem Benlaifa 2,Radia Buasla 3, Malika Berredjem 3, Rafika Bardi 4

1 University of Badji Mokhtar, Department ofBiochemistry, Laboratory of Applied Bio-chemistryand Microbiology, Annaba, Algeria2 University of Badji Mokhtar, Department of Biology,Laboratory of Cell Toxicology, Annaba, Algeria3 University of Badji Mokhtar, Laboratory of AppliedOrganic Chemistry, Synthesis of Biomolecules andMolecular Modelling Group, Annaba, Algeria4 Laboratory of Immunology, EPS Charles Nicolle,Tunis, TunisiaE-mail address: h [email protected]

Toxoplasma gondii (T. gondii) is a significant cause of congeni-tal disease and an important AIDS opportunistic pathogen. Currentavailable drugs do not give satisfactory results and often haveonly a static and several adverse side effects. The need to developand evaluate new drugs is critical. The purpose of this study is toinvestigate the in vivo effect of two new chiral N-acylsulfonamidebis-oxazolidin-2-ones (BOSP and BOSV) on T. gondii’s infectedSwiss mice. The anti-T. gondii RH strain activities of the newmolecules were investigated in mice after molecules treatment atdifferent concentrations by using a real-time PCR-based assay forthe detection and quantification of the parasite. Oligonucleotideprimers and a fluorescence-labeled TaqMan probe were used to
amplify the T. gondii B1 gene. Toxoplasma-specific DNA was demon-strated in all samples from infected mice. The parasite quantityshowed that BOSP molecule has better effect (0.78 × 103 ± 0.27)
dx.doi.org/10.1016/j.bdq.2017.02.120



dx.doi.org/10.1016/j.bdq.2017.02.121



dx.doi.org/10.1016/j.bdq.2017.02.122



S lar Det

ttauidisti

h

PApi

DB

1

2

BE

T(tfcmco

fwdTadctsqc

civmpecdrafd41ri1o

appear to be compatible with their inhibitory role in the control ofovarian angiogenesis



han BOSV one (1.83 × 103 ± 0.19) but did not completely inhibithe parasite proliferation. The molecules inhibited the parasite in

dose dependent manner. The intensity of amplification prod-cts increased (3.85 × 103 ± 0.31) when treatment started late after

nfection. These findings suggest continuous parasite replicationespite the treatment. In conclusion, our results showed a promis-

ng treatment effect of the tested molecules. Otherwise, the presenttudy revealed that concentration and duration of tested moleculesreatment are major factors that influence the course of Toxoplasmanfection in infected mice.


O 35quantitative real-time PCR for

harmacokinetic analysis of themmunotherapeutic TLR9 agonist lefitolimod

etlef Oswald 1,∗, Kerstin Kapp 1,urghardt Wittig 2, Manuel Schmidt 1

MOLOGEN AG, GermanyFoundation Institute Molecular Biology andioinformatics, Freie Universität Berlin, Germany-mail address: [email protected]

Background: Lefitolimod (MGN1703) is a synthetic DNA-basedLR9 agonist that serves as an immune surveillance reactivatorISR) in immune-oncology and infectious diseases. It is currentlyested in several clinical trials, of which a pivotal phase III trialor first line maintenance treatment of patients with metastaticolorectal carcinoma is the most advanced. To analyze the phar-acokinetic (PK) profiles in different species during preclinical and

linical development processes a hydrolysis probe qPCR was devel-ped and successfully employed.

Methods: A hydrolysis probe qPCR was developed and validatedor lefitolimod, a dumbbell-shaped covalently-closed moleculeith two identical single-stranded loops linked by a palindromicouble-stranded stem and an overall size of only 116 nucleotides.o avoid variation in efficacy of sample preparation due to the shortnd covalently-closed DNA molecule, serum or blood samples ofifferent species were used directly for qPCR without any purifi-ation or extraction steps. The small size of lefitolimod but alsohe identical single-stranded loops connected by a double-strandedtem with a palindromic sequence posed difficult challenges forPCR. In addition possible negative influences from serum or bloodomponents had to be avoided.

Results: Despite the above-described challenges a reliable assayould be developed and validated. PK measurements of lefitolimodn animal toxicity studies and a phase 1 clinical trial with healthyolunteers (MGN173-C4) were performed. The quantitative deter-ination of lefitolimod was possible with high intra-assay

recision and accuracy. The inter-assay precision and accuracy wasstablished by analyzing 6 QC sets together with a complete set ofalibration standards at 3 different days. The results show that theetermination of lefitolimod is possible with high inter-day accu-acy and precision as well. Linearity is given between 1 × 10−10 g/�lnd 1 × 10−14 g/�l (100–0.01 ng/ml). In human serum samplesrom the MGN1703-C04 trial standard PK parameters could beetermined. The mean Cmax was 189 ± 101 ng/mL (range: 53.4 to49 ng/mL) and the mean AUC0–t was 5001 ± 2518 ng hr/mL (range:264–10,984 ng hr/mL). Although there was a moderately wide
ange in the Cmax and AUC0–t values, the Tmax and t1/2 were sim-lar for all subjects. Tmax ranged from 8 to 16 h, with a median of4 h. The values for t1/2 ranged from 9.36 to 17.9 h, with a medianf 12.7 h.

Conclusions: The data show that the hydrolysis probe qPCRmethod is able to analyze PK parameters of the short, dumbbell-shaped and covalently-closed DNA-based TLR9 agonist lefitolimodfrom serum or blood samples of different species during preclinicaland clinical development.


PO 36Expression and localization of thethrombospondin family members duringcorpus luteum formation and function in thebovine ovary

Bajram Berisha 1,2,∗, Dieter Schams 2,Daniela Rodler 3, Fred Sinowatz 3,Michael W. Pfaffl 2

1 Faculty of Agriculture and Veterinary, University ofPrishtina, Prishtinë, Kosovo2 Institute of Animal Physiology and ImmunologyWeihenstephan, Technical University of Munich,Freising, Germany3 Institute of Anatomy, Histology and Embryology,Ludwig Maximilian University of Munich, Munich,GermanyE-mail address: [email protected]

The objective of this study was to characterize the expressionpatterns and localization of the thrombospondin family members(THBS1, THBS2) and their receptors (CD36 and CD47) in bovinecorpus luteum (CL). First, the corpus luteum (CL) was assignedto the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and>18 of the estrous cycle and of pregnancy (months 1–2, 3–4, 6–7and >8). Second, the corpora lutea were collected by transvaginalovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after induc-ing luteolysis by injecting a prostaglandin F2alpha (PGF) analog.The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization byimmunohistochemistry. The mRNA expression of THBS1, THBS2,and CD47 in the CL during the estrous cycle was high, but decreasedsignificantly during pregnancy. After induced luteolysis, throm-bospondins increased significantly to reach the maximum level at12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporalexpression and localization pattern of the thrombospondins andtheir specific receptors in the bovine corpora lutea during the differ-ent physiological phases of the estrous cycle and induced luteolysis

dx.doi.org/10.1016/j.bdq.2017.02.123



dx.doi.org/10.1016/j.bdq.2017.02.124



dx.doi.org/10.1016/j.bdq.2017.02.125

lar Det

PGeo

VS

1

A2

DE

oosbemiceecmutp4wddyegeac(eci2tIoiIvtmeIl(fiet

h


O 37ene expression signatures of bovine in vitrombryos produced at the air-liquid interphasen oviduct epithelial cells

era A. van der Weijden 1,∗, Shuai Chen 2,usanne E. Ulbrich 1, Jennifer Schoen 2

ETH Zurich, Animal Physiology, Institute ofgricultural Sciences, SwitzerlandLeibniz Institute for Farm Animal Biology, FBNummerstorf, Germany-mail address: [email protected]

Ongoing development in the field of in vitro culture (IVC) meth-ds for embryos is driven by increasing knowledge of the effectsf IVC conditions on offspring. Besides morphological alterations,everal studies have shown aberrant gene and protein expressiony comparing in vivo and in vitro developed embryos. A novelmbryo culture system where embryos are co-cultured without IVCedium on differentiated bovine oviductal epithelial cells (BOEC)

s hypothesised to resemble more closely the in vivo situation thanonventional IVC. In the present study, we evaluated the genexpression of development-related genes to analyse the effect ofmbryo co-culture with BOEC. Embryos developed under serum-ontaining in vitro conditions (IVC-S), in a commercial serum-freeedium (IVC-SF), or in a co-culture system with BOEC (IVC-BOEC)

ntil the 8-cell (n = 3 pools of five embryos per group) and the blas-ocyst stage (n = 6 per group), respectively. The gene expression ofools of 8-cell embryos, and single blastocysts was assessed using a8.48 Dynamic ArrayTM on a Biomark HD instrument. Target genesere selected based on published transcriptomics and RT-qPCRata of developing bovine embryos. Clustering of the embryos byevelopmental stage was confirmed by principle component anal-sis, where there was no indication of a culture condition-inducedffect. At each developmental stage, nine differentially expressedenes were detected by comparing IVC-S, IVC-SF, and IVC-BOECmbryos. At the 8-cell stage, the expression of CDH1 and NOS2 (4.8-nd 1.8-fold upregulated in IVC-BOEC vs IVC-S, respectively) indi-ates an interaction of embryos with BOEC. The expression of APEX12.1-fold upregulated in IVC-SF vs IVC-S) demonstrates the adverseffect of culturing embryos under serum conditions. This is in con-ordance with the expression of BAX (2.5- and 3.2-fold upregulatedn IVC-BOEC vs IVC-S and IVC-SF, respectively), PLAGL1 (3.4- and.4-fold upregulated in IVC-BOEC vs IVC-S and IVC-SF, respec-ively), and SMPD2 (3.1-and 2.8-fold downregulated in IVC-SF vsVC-S and IVC-SF, respectively). Blastocysts also displayed supportf the BOEC, given the expression of CCL26 (2.6-fold upregulatedn IVC-BOEC vs IVC-S), CDH1 (2.1-fold upregulated in IVC-BOEC vsVC-SF), and NID2 (4.4- and 6.5-fold downregulated in IVC-BOECs IVC-S and IVC-SF, respectively). Similar to the 8-cell embryos,he serum conditions altered gene expression of genes involved in

etabolism. In the blastocysts, this is reflected by differential genexpression of GLUT-5 (2.3- and 2.7-fold downregulated in IVC-SF vsVC-S and IVC-SF, respectively), SREBP1 (1.9- and 2.3-fold upregu-ated in IVC-BOEC vs IVC-S and IVC-SF, respectively), and CYP51A13.2-fold downregulated in IVC-BOEC vs IVC-S). Collectively, thendings show distinct gene expression patterns in response to the
nvironment of the early developing embryo, underlining the plas-icity of the embryo during this critical developmental phase.


PO 38Deregulated genes in hematopoietic stem cellsisolated from spleen and peripheral blood ofpatients with myelofibrosis

Paolo Catarsi 1,∗, Francesca Cordero 2,4,Giulio Ferrero 2,3,4, Marco Beccuti 4,Valentina Poletto 1, Elisa Bonetti 1, Laura Villani 1,Margherita Massa 5, Gabriela Fois 1,Rita Campanelli 1, Umberto Magrini 1,Vittorio Rosti 1, Giovanni Barosi 1

1 Center for the Study and Treatment ofMyelofibrosis, Biotechnology Research Area,Fondazione IRCCS Policlinico “San Matteo”, Pavia,Italy2 Center for Molecular Systems Biology, University ofTurin, Turin, Italy3 Department of Clinical and Biological Sciences,University of Turin, Turin, Italy4 Department of Computer Science, University ofTurin, Turin, Italy5 Biotechnology Research Area, Fondazione IRCCSPoliclinico “San Matteo”, Pavia, ItalyE-mail address: [email protected]

An important issue in myeloproliferative diseases researchis to test the hypothesis that the neo-angiogenesis, which isobserved in spleen and bone marrow, involves endothelial cellsderived from the myeloid, neoplastic clone (Rosti V. et al., Blood2013). In this study we analyzed the gene expression profile inCD34+ hematopoietic cells from the spleen and peripheral bloodof patients with myelofibrosis (MF) and healthy individuals (HI).The analyzed transcripts were selected from literature amongthose that characterize the neoangiogenic gene signature and thoseinvolved in deregulated expression pathways in MF.

By using quantitative reverse transcription-PCR, we measuredthe expression levels of 141 targets and 5 reference genes, in CD34+cells isolated by immunomagnetic separation from the spleen andperipheral blood of 4 patients and 3 HI. The same genes were alsoanalyzed in circulating endothelial progenitor cells (EPCs), whichwere isolated from peripheral blood of 4 MF patients and 4 HI andsubsequently expanded in vitro. The lists of differentially expressedgenes were analyzed through several tools (i.e. STRING, ENRICHRand GSEA).

The most significant upregulated genes in CD34+ splenic cellsfrom patients, included transcripts (GATA1, HBB, TAL1, GATA2,PTGS1) belonging to the molecular signature of CD34+ cells iso-lated from the bone marrow (BM) of patients with chronic myeloidleukemia (Diaz-Blanco E. et al., Leukemia 2007). The genes GATA1,TAL1, ITGB3, GATA2 and PF4 were also found overexpressed in astudy designed to characterize the genes essential to the devel-opment of megakaryocytes (Tenedini E. et al., Blood 2004). Thesetranscripts are part of an expression pattern characteristic forimmature stem cells as well as megakaryocyte-erythrocyte progen-itor cells. Another group of transcripts overexpressed in patientsspleens, which includes some of the above mentioned genes (CD34,ANGPT1, PF4, GATA2, PTGS1), has been observed in a comparisonbetween circulating CD34+ cells from patients with MF, and CD34+cells isolated from the bone marrow of HI (Guglielmelli P. et al.,Stem Cells 2007). Moreover, in partial disagreement with the initialhypothesis, a group of genes implicated in vascular development(FLT1, THBS1, FN1, CTGF, IL8, EPHA2, PROK2, LOXL2) were found sig-
nificantly underexpressed in CD34+ cells isolated from the spleenof patients compared to HI. For these reasons, we analyze the geneexpression pattern in EPCs. These cells, although not belongingto the mutated clone, express hematopoietic specific transcripts


dx.doi.org/10.1016/j.bdq.2017.02.126



S lar Det

leg

ptt

h

PAd

RWS

1

C2

E

atHlteciD

uitaqt

wViqwR[e

cattgba(ncttwR

[

[[

[


inked to immunity and inflammation. We observed that the dis-ase modifies their phenotype by increasing the expression of someenes, including PTGS1.

Our results point toward a model in which, in the spleen ofatients with MF, an altered hematopoietic stem cell differentia-ion could induce an inflammation-mediated angiogenesis throughhe overexpression of PTGS1 in cells of the splenic tissue.


O 39microbead-based assay for the real-time

etection of multiple HPV genotypes

oy-Arne Senkel 1, Claudia Deutschmann 1,erner Lehmann 2, Peter Schierack 1,

tefan Rödiger 1,∗

Brandenburg University of Technologyottbus–Senftenberg, Senftenberg, GermanyAttomol GmbH, Lipten, Germany-mail address: [email protected]

Background: Human papillomaviruses (HPV) infect skin cellsnd mucous membranes and usually cause a local cell prolifera-ion after a successful infection. An infection with low-risk typePV leads in most cases to benign proliferations such as epithe-

ial cysts or skin warts. High-risk types cause lesions that may leado the progression of malignant tumors (e.g., cervical cancer). Oth-rs have shown that HPV-DNA can be found in about 90% of allervical carcinomas. In current diagnostics, viral DNA is detectedn patient samples using the polymerase chain reaction (PCR) orNA-hybridization assays.

Objective A multiplex quantitative PCR system, which can besed to determine the type and approximate viral load level of HPV

nfections might be a valuable tool in diagnostics and fundamen-al research. We aimed to develop a multiplex quantitative PCRssay based on microbead encoded targets for the detection anduantification of HPV. The possible benefits are an increased samplehroughput while reducing costs and effort.

Methods A primer/probe system for the detection of HPVas established for the microbead-based quantitative PCR of theideoScan platform. Negative and positive controls were integrated

nto the assay to improve the reliability. Furthermore, the absoluteuantification of HPV copies in patient samples was accomplishedith the QX200 Droplet DigitalTM PCR (ddPCR) system from Bio-ad

®. All data were analyzed using the chipPCR [1] and MBmca

2] packages for the R statistical computing language as describedlsewhere [3,4].

Results and conclusion: For the HPV detection, a shortonserved region (65 bp) of the L1 gene was appropriate. Anbsolute quantification of virus copies was made ahead ofhe assay establishment through a ddPCR system. The quan-ification and differentiation of the HPV 16 and HPV 18enotypes was possible on the basis of sequence differencesetween specific hydrolysis probes, both in the liquid phasend on the microbead-surface. An internal amplification controlHypoxanthin-Guanin-Phosphoribosyltransferase 1) and an exter-al extraction control (�-globin) were integrated in the assay andould be used as reliable assay controls (100% recovery rate). Withhis assay it was feasible to detect five different targets in one reac-ion. Since we used two distinct microbead populations per target
e achieved a quasi ten-plex assay.eferences
1] S. Rödiger, M. Burdukiewicz, P. Schierack, Bioinformatics 31 (2015) 2900–2902,http://dx.doi.org/10.1093/bioinformatics/btv205.


2] S. Rödiger, A. Böhm, I. Schimke, R J. 5 (2013) 37–53.3] S. Rödiger, M. Burdukiewicz, K.A. Blagodatskikh, P. Schierack, R J. 7 (2015)

127–150.4] A.-N. Spiess, C. Deutschmann, M. Burdukiewicz, R. Himmelreich, K. Klat, P.

Schierack, S. Rödiger, Clin. Chem. 61 (2015) 379–388, http://dx.doi.org/10.1373/clinchem.2014.230656.


PO 40VitiPatho: A qPCR-method for the prediction ofthresholds for fungizide application

Bernhard Setzer 1,∗, Rene Fuchs 2

1 BS-diagnostik, Germany2 State Institute for Viticulture and Oenology ofFreiburg, GermanyE-mail address: [email protected]

Plasmopara viticola is a strictly biotrophic oomycete that causesdowny mildew, which is one of the most important grapevine dis-eases. Control of the disease is most often achieved by fungizideapplication, which may have severe environmental consequences.To optimize the use of fungizide the “State Institute for Viticul-ture and Oenology of Freiburg” is offering predictive meteorologicaldata via a system called “VitiMeteo”. In order to obtain insight intothe dynamic of host responses to grapevine downy mildew (Plas-mopara viticola), we in addition developed a monitoring systemfor air-borne fungal pathogens “VitiPatho”. Spores of Plasmoparaviticola were sampled using a modified microbiological air samp-ling system MBASS30. 100 L of sampled air were collected directlyinto a 200 �l PCR tube containing lysis buffer. Samples were thenstored on ice for further treatment in the lab. After lysis sampleswere diluted and specific target sequences were amplified by qPCRusing a Taq-polymerase from myPOLS, kindly provided by RamonKranaster. The PCR-reaction was checked for specificity and lin-earity. Relative quantification was calculated on the basis of theobtained ct-values during the growing season in 2016.

Results:

1. Peronospora viticola infection could be detected as early as endof May (5 to 6 leaves) on the vineyard “Wohnhalde”.

2. The onset of Peronospora viticola infection on the vineyard of the“Lorettoberg” was about two weeks later in comparison to thelocation “Wohnhalde”.

3. Measured DNA from collected air samples of Peronospora viticolaincreased during infection by a factor of 1000.

4. During the Peronospora infection, the protection of plants bydifferent fungizide could be visualized by low signals of Per-onospora viticola DNA compared to the infected plants at thesame vineyard investigated.


PO 41A novel high-multiplex homogeneous PCR assayformat

Søren Morgenthaler Echwald

Anapa Biotech A/S, Denmark
E-mail address: [email protected]
Multiplex PCR has become an increasingly popular method toprovide more, clinically relevant answers from the same sample. In

dx.doi.org/10.1016/j.bdq.2017.02.127




















dx.doi.org/10.1016/j.bdq.2017.02.128



dx.doi.org/10.1016/j.bdq.2017.02.129



lar Det

mscabbchr

aboaan

di

h

h

PDfP

FFP

E

EEmaa

ocate

op

btsaL0

caa

groups, new biomarkers are necessary for assessing genetic char-acteristics of lysosomal storage disorders in different populations.



any clinical settings, achieving multiplex answers from the sameample provide benefits both in terms of cost, speed, added clini-al value as well as preservation of limited samples. In cases suchs sepsis, RSV-testing, gastrointestinal testing and many others, aroad spectrum of agents are relevant for testing to assess possi-le infections. However, PCR readout is commonly limited to theurrent maximum of 4–5 fluorophores on most instruments. Weave developed MeltPlex – a homogenous assay method to allowead-out of more than 20 answers from a single PCR reaction.

Material/methods: MeltPlex utilizes a system of labelled probesllowing each to be read out by subsequent melting curve analysisy more than 5 probes per fluorophore. By utilizing meltcurve read-ut of modified probes – one for each target – rather than the onlymplicons, the system adds an extra level of specificity to meltcurvenalysis. Reaction and melting analysis is performed without theeed to re-open PCR reaction tubes.

Results: We will present proof-of-principle of the method andemonstrate its relevance in routine clinical infectious agent test-

ng.Conclusions: MeltPlex comprise a robust, high-multiplex,

omogeneous system to provide 20+ readouts per PCR reaction.


O 42etection of EGFR mutations in cell-free DNA

rom patients affected by lung cancer:reanalytical and analytical aspects

rancesca Salvianti ∗, Francesca Malentacchi,rancesca Damiani, Irene Mancini, Mario Pazzagli,amela Pinzani

Scienze Biomediche, Sperimentali e Cliniche, Italy-mail address: [email protected]

Lung cancer is the leading cause of cancer death worldwide.GFR mutations are driver oncogenic mutations against whichGFR tyrosine kinase inhibitors (TKIs) represent an effective treat-ent. Recently AIOM guidelines indicated cell-free DNA (cfDNA) assurrogate for the determination of EGFR status in patients withoutvailable tumor sample.

Our aim is to evaluate different approaches for the identificationf EGFR mutations in cfDNA from lung cancer patients in view of alinical application. In particular we focused on mutations associ-ted with sensitivity to EGFR TKIs, such as deletions in exon 19 andhe mutation p.L858R in exon 21, and on the mutation p.T790M inxon 20, responsible for resistance to treatment.

We considered the preanalytical phase by comparing two meth-ds, manual and automated, for cfDNA extraction: the automaticrocedure showed a better yield than the manual.

We compared the analytical performances of two methodsased on real-time quantitative PCR (qPCR) with those of a digi-al PCR (dPCR) approach. From the results obtained on referencetandard samples at known percentages of mutated alleles dPCRppeared as the most sensitive method, being capable of detecting858R mutation and a deletion in exon 19 in a sample containing.1% mutated DNA.

Preliminary data on patients affected by lung cancer showedoncordant results between the two qPCR methods. Some discrep-ncies in the mutational status of EGFR were found between tissuend cfDNA.


Upon optimization and standardization of methods, non-invasive analysis of cfDNA might replace biopsy for a precisionmedicine approach in lung cancer.


PO 43Allelic frequency of a 24-bp duplication in exon10 of the CHIT1 gene in the general Iranianpopulation by HRM analysis

Behrooz Motlagh 1,∗, Mohammad Taghikhani 2,Nasim Abedimanesh 3

1 Zanjan University of Medical Sciences, IslamicRepublic of Iran2 Tarbiat Modares University, Islamic Republic of Iran3 Tabriz University of Medical Sciences, IslamicRepublic of IranE-mail address: [email protected]

Background: The human chitinase chitotriosidase enzyme,which is encoded by the CHIT1 gene, is produced by macrophagesand may be important in immune responses to chitin-containingorganisms, such as fungi. Plasma chitotriosidase activity is used todiagnose and monitor some forms of lysosomal storage disorders,such as Gaucher disease. This metabolic disorder is an autosomalrecessive lysosomal storage disease. Clinical manifestations of thedisease include hematological and skeletal complications includingbone pain (BP) and bone crisis, medullary expansion, osteope-nia, osteolysis, osteonecrosis, and pathological fractures. However,homozygous duplication of a 24-bp region in exon 10 of the CHIT1gene eliminates enzyme activity and may complicate disease mon-itoring. The high prevalence of this mutation highlights the needto determine its frequency in different populations and screenpatients for this mutation in order to verify whether chitotriosi-dase activity is a reliable marker of lysosomal storage disease. Thisstudy investigated the allele frequency of the 24-bp duplication inthe general Iranian population.

Methods: To identify the 24-bp duplication in exon 10 of theCHIT1 gene (H allele), genotyping of DNA extracted from peripheralblood leukocytes of 577 healthy Iranians was performed using PCRamplification and High Resolution Melting (HRM) PCR techniques.

Results: In this study, heterozygous and homozygous dupli-cations were detected in 183 (31.7%) and 35 (6.1%) subjects,respectively. In addition, the allelic frequency was 21.9% (95% con-fidence interval).

Conclusion: Our study indicates that genotype analysis byHRM-PCR is a fast, reliable, and highly accurate screening approachfor identifying the 24-bp duplication in CHIT1 exon 10. Due tothe wide range of duplication frequencies among different ethnic

dx.doi.org/10.1016/j.bdq.2017.02.130



dx.doi.org/10.1016/j.bdq.2017.02.131



dx.doi.org/10.1016/j.bdq.2017.02.132

S lar Det

PDmM

KMS

1

2

U3

H4

oGE

mtImessaa(

lmatafi

pmRefhfiistbdaap

fMtTpR

[

[

[

[[2] Spiess, et al., BMC Bioinform. 9 (April) (2008) 221.[3] P. Buchwald, Math. Biosci. 205 (January (1)) (2007) 108–136.


O 44evelopment of an R package for qPCR dataanagement with the real-time PCR Dataarkup Language

onstantin Blagodatskikh 1,ichał Burdukiewicz 2, Andrej-Nikolai Spiess 3,

tefan Rödiger 4,∗

Evrogen JSC, Moscow, RussiaDepartment of Genomics, Faculty of Biotechnology,niversity of Wrocław, Wrocław, PolandUniversity Medical Center Hamburg-Eppendorf,amburg, GermanyInstitute of Biotechnology, Brandenburg Universityf Technology Cottbus–Senftenberg, Senftenberg,ermany-mail address: [email protected]

Objective: Real-time quantitative PCR (qPCR) is one of theost widely applied methods in molecular biology, diagnos-

ics, forensics and genetical testing. The guideline for Minimumnformation for Publication of Quantitative Real-Time PCR Experi-

ents (MIQE) [1] was established to facilitate the comparison ofxperimental results obtained from qPCR. The MIQE guidelinesuggest the Real-time PCR Data Markup Language (RDML) as thetandard qPCR interchange data metadata and values betweenpplications [2]. RDML is a vendor independent and freely avail-ble file format. It is based on the eXtensible Markup LanguageXML).

Aim and methods: The cross-platform statistical computinganguage R is the de facto standard in applied statistical bioinfor-

atics, provides comprehensive tool-sets for reproducible researchnd is suitable for standalone desktops or servers. Althoughhere are R packages for qPCR and melting curve analysis avail-ble, it was not previously possible to seamlessly process RDMLles [3].

Results: We developed the cross-platform open source RDMLackage for the statistical computing language R. RDML is compli-ent to RDML ≥ v. ∼1.2 and provides functionality to (i) import

DML and native data from various PCR machines vendors; (ii)xtract sample information (e.g., targets, concentration); (iii) trans-orm data to various formats of the R environment; (iv) generateuman readable experiment summaries; and (v) to create RDMLles from user data. In addition, RDML offers a graphical user

nterface, designated rdmlEdit, to edit RDML files and performimple manipulation of the PCR curves like background subtrac-ion and Cq calculation. rdmlEdit can be used as tool from arowser or integrated development environments such as RStu-io. RDML has import functionality for additional file formats suchs comma separated values. Since the software is based on R, allvailable packages can be combined with function from the RDMLackage.

Conclusion: The RDML package is a tool that can be usedrom the command-line to design advanced analysis pipelines.

oreover, the implemented graphical user interface enableshe users to perform rapid management of their qPCR data.he RDML package is available at https://cran.r-project.org/ackage=RDMLeferences

1] S.A. Bustin, et al., The MIQE guidelines: minimum information for publicationof quantitative real-time PCR experiments, Clin. Chem. 55 (2009) 611–622,
http://dx.doi.org/10.1373/clinchem.2008.112797.
2] S. Lefever, J. Hellemans, F. Pattyn, D.R. Przybylski, C. Taylor, R. Geurts, A.Untergasser, J. Vandesompele, on behalf of the R. Consortium, RDML:structured language and reporting guidelines for real-time quantitative PCR

[[[


data, Nucl. Acids Res. 37 (2009) 2065–2069, http://dx.doi.org/10.1093/nar/gkp056.

3] S. Rödiger, M. Burdukiewicz, K.A. Blagodatskikh, P. Schierack, R as anenvironment for the reproducible analysis of DNA amplification experiments, RJ. 7 (2015) 127–150.


PO 45A comparison of nonlinear fitting methods toraw qPCR amplification curves: Performance,caveats and implications for Cq and E estimation

Andrej-Nikolai Spiess 1,∗, Stefan Rödiger 2,Joel Tellinghuisen 3

1 Department of Andrology, University HospitalHamburg-Eppendorf, Germany2 Brandenburg University of TechnologyCottbus-Senftenberg, Senftenberg, Germany3 Department of Chemistry, Vanderbilt University,Nashville, TN 37235, USAE-mail address: [email protected]

In the last decade, several algorithms have been developedfor the estimation of quantification cycles (Cq) and amplifica-tion efficiencies (E) that employ whole-curve fitting of the qPCRamplification trajectory (compiled in [1]). In the majority of cases,this is conducted by ad-hoc fitting of four- or five-parameter sig-moidal models (e.g. logistic, log-logistic, Boltzmann, Richards) thatcan or cannot account for curve asymmetry around the “pointof inflection” [2]. However, the scientific literature provides aplethora of nonlinear models that have not found their way intoqPCR analysis or are not yet implemented in routine software,such as six-parameter (incorporating linear baseline shift) andseven-parameter (incorporating quadratic baseline shift) models,linear-exponential (Spiess, unpublished) and bilinear-exponential[3] hybrid models, as well as recursive growth models [4]. Non-parametric alternatives also exist, such as exact or smoothingsplines [5]. In the application of these models, it is essential touse Information Criteria (AIC, BIC) to estimate the goodness-of-fit, as the increasing number of parameters inherently results inimproved (over-)fitting [6]. In addition to standard nonlinear fit-ting, weighted fitting can dramatically improve fit quality, becausethe increasing dispersion of data points in relation to their mag-nitude (heteroscedasticity) can be compensated by giving lessfitting weight to curve regions displaying high variance, e.g. base-line and plateau phase. This weighting compensation can also beincluded in the fitting process by iteratively estimating the opti-mal weighting parameters (generalized nonlinear models). All theabove methods employ nonlinear least-squares, which minimizesthe sum-square of vertical residuals. Along these lines, we alsoinvestigate the rarely employed but highly important method oforthogonal nonlinear least squares [7] which exhibits optimal fit-ting in the steep slope region of the qPCR curves by minimizingorthogonal residuals. For all these methods, we show their per-formance, advantages and drawbacks on high-numbered technicalreplicate datasets, especially with respect to the elimination ofsystem-intrinsic Cq value periodicities previously identified by us[8].References1] Ruijter, et al., Methods 59 (January (1)) (2013) 32–46.

4] Carr, Moore, PLoS ONE 7 (5) (2012) e37640.5] Spiess, et al., Clin. Chem. 61 (February (2)) (2015) 379–388.6] Spiess, Neumeyer, BMC Pharmacol. 10 (June) (2010) 6.



https://cran.r-project.org/package=RDML















dx.doi.org/10.1093/nar/gkp056








dx.doi.org/10.1016/j.bdq.2017.02.133



lar Det

[[

h

PTutaw

J

E

qgmFfu�oKg

h

PUpc

JM

1

2

E

MaNatyawwaoo

scipmmipp


7] Boggs, et al., SIAM J. Sci. Stat. Comput. 8 (6) (1987) 1052–1078.8] Spiess, et al., Sci. Rep. 6 (December) (2016) 38951.


O 46he role of amplification efficiency and itsncertainty in assessing relative expression byhe ��Cq method: A nonlinear least-squarespproach that overcomes deficiencies in aidely used SAS program

oel Tellinghuisen

Vanderbilt University, United States-mail address: [email protected]

Relative expression ratios are commonly estimated in real-timePCR studies by comparing the quantification cycle for the targetene with that for a reference gene in the treatment samples, nor-alized to the same quantities determined for a control sample.

or the “standard curve” design, where data are obtained for allour of these at several dilutions, nonlinear least squares can besed to assess the amplification efficiencies (AE) and the adjusted�Cq and its uncertainty, with automatic inclusion of the effect

f uncertainty in the AEs. A simple algorithm is illustrated for thealeidaGraph program and used to illustrate flaws in an SAS pro-ram that has been used for this purpose in the last decade.


O 47rine microRNAs as potential diagnostic andrognostic biomarkers of urinary bladderancer

aroslav Juracek 1,2, Sona Klusova 1,∗,ichal Stanik 2, Jan Dolezel 2, Ondrej Slaby 1,2

Central European Institute of Technology, CzechiaMasaryk Memorial Cancer Institute, Czechia-mail address: [email protected]

Bladder cancer is the most common cancer of the urinary tract.ore than 90% of bladder cancers are urothelial carcinoma, which

re divided into non-muscle-invasive and muscle-invasive forms.on-muscle-invasive tumors frequently recur (50–70%) and canlso progress to invasion form (10–15%). These patients are moni-ored by cystoscopy and may have multiple resections over manyears. Improved monitoring method is needed, ideally via urinenalysis, which could reduce the morbidity and costs associatedith long follow up. Currently there are no molecular biomarkershich could diagnose or accurately predict disease progression. We

imed to develop a clinically applicable, specific and sensitive panelf urine microRNAs enabling detect bladder cancer and predict riskf progression to muscle-invasive form.

Within the exploratory phase of study we have analyzed expres-ion profiles of 1733 miRNAs in urine supernatant of 16 bladderancer patients (6 muscle invasive, 5 high-grade muscle non-nvasive, 5 low-grade muscle non-invasive), 17 controls, 10 RCCatients and 4 urinary tract infections (UTI) using AffymetrixiRNA microarrays. Diagnostic and prognostic potential of selectedicroRNAs was further validated on independent samples in train-

ng phase (50 bladder cancer patients, 15 controls) and validationhase (100 bladder cancer patients, 55 controls, 45 renal canceratients) using specific TaqMan assays and qRT-PCR method.


Global expression profiling identified set of 76 miRNAs able dis-tinguish bladder cancer patients from healthy controls (P < 0.01),thereof 64 highly up-regulated and 12 down-regulated. Moreover23 miRNAs were able distinguish invasive and non-invasive formsof UCUB (P < 0.01) and 18 miRNAs high-grade and low-grad non-invasive (P < 0.01). Set of 12 miRNAs with highest expression leveland statistical significance was validated in training phase of study.Based on the results the panel of three miRNAs (miR-31, miR-93,miR-191) was profiled. In validation phase we confirmed diagnosticpotential and ability of this urine miRNA-based panel to diagnosepatients with bladder cancer with high sensitivity and specificity(AUC = 0.8794, sensitivity = 82%, specificity = 80%).

Our data have shown that urinary microRNAs could serve as sen-sitive and specific biomarkers of urinary bladder cancer and couldbe useful tool to increase sensitivity of standard cytological exam-ination and reduce costs associated with long-term follow-up ofbladder cancer patients. This work was supported by Ministry ofHealth of the Czech Republic, grant nr. 15-31071A, 15-34678A andCEITEC 2020 (LQ1601). All rights reserved.


PO 48Is quantitative microRNA expression profilingusing Fluidigm microfluidic dynamic arraysreliable? Taqman microRNA assay evaluation

Vlasta Korenková 1,∗, Jan Král 2,Veronika Vymetálková 2, Vendula Novosadová 1,Lucie Langerová 1, Pavel Vodicka 2, Jana Slysková 2

1 Institute of Biotechnology, Czechia2 Institute of Experimental Medicine, CzechiaE-mail address: [email protected]

High-throughput profiling methods are being used to savebiological material, time and hopefully also money. During ourexperiment we used two high-throughput instruments to measuremicroRNA expression: 3D-Gene (Toray) for a discovery phase ofrectal cancer biomarkers search and BioMark, a high-throughputqPCR instrument from Fluidigm, for the validation phase. Duringthe initial search for a reliable microRNA system, we excludedExiqon platform due to its exclusive use of SYBR green chemistry.The SYBR green is not compatible with microfluidic instruments.There were several articles supporting use of Taqman microRNAsystem together with BioMark, therefore we decided to take thisdirection.

Here we describe how we evaluated probes from Life Tech-nologies to be reliable for BioMark. Twenty patients were sampledfor rectal tumour tissue and adjacent healthy rectal tissue. Using3D-Gene microRNA array, 2000 microRNA were screened for differ-ential expression. Seventy-one candidate microRNAs were selectedfor next, validation, phase of experiment. None or very late ampli-fication (Cq > 45) was detected using 23 Taqman microRNA probes.There might be two reasons for the lack of amplification. One isthat the probe does not work, which was tested by temperaturegradients. The other reason can be limited correlation betweenmicroarray system and qPCR that is reported in the literature. Thecorrelation can differ from 0.48 to 0.94. Other 13 assays providedvery late amplification (Cq > 36). All these mentioned probes wereexcluded from further validation.

The second half of Taqman microRNA assays were evalu-
ated. The assay precision, efficiency, limit of quantification weremeasured. Because only limited amount of biological materialcan be used for high-throughput experiment, pre-amplificationis required. This additional step was also evaluated. Finally, we
dx.doi.org/10.1016/j.bdq.2017.02.134



dx.doi.org/10.1016/j.bdq.2017.02.135



dx.doi.org/10.1016/j.bdq.2017.02.136



S lar Det

epm

Tvgq

0

h

PAG

JS

E

dos(hwoivaamdi

amsirqsitiBaipbss3Ftpmciiim

doubling the high-throughput profiling capacity by RT-qPCR from760 to 1300 miRNAs.



nded up with several variables for each assay that had to be com-ared. Principal component analysis helps us to identify 21 reliableicroRNA assays.In summary, the assay evaluation revealed that 70% of obtained

aqman microRNA assays could have not been used for BioMarkalidation phase. However, 21 approved assays (30%) enabled us toet consistent and reliable results that were obtained from subse-uent experiments.

This project was funded by the Czech grant agency GACR: GA15-8239 S.


O 49nalysis of microRNA isoforms (isomiRs) inalaxy and their differences in expression levels

ochen Bick ∗, Susanne E. Ulbrich,tefan Bauersachs

ETH Zurich, Switzerland-mail address: [email protected]

In general, the analysis of small RNA-Sequencing (RNA-Seq)ata is more challenging compared to standard RNA-Seq. More-ver, an additional problem when working with livestock species,uch as the pig, is that the annotation of small non-coding RNAsncRNAs), e.g., microRNAs (miRNAs), is far from being complete. Inumans, a great variety of ncRNAs including miRNAs are known,hich can be used as orthologue information for the annotation

f sequences derived from other mammalian species. This helps toncrease the percentage of annotated miRNA sequences and theirarious isomiRs. IsomiRs are miRNA variants that differ at the 3′

nd/or 5′ end, which results from imprecise and alternative cleav-ge during the pre-miRNA processing and post-transcriptionalodifications represented by added or deleted nucleotides. These

ifferent isomiRs can exhibit different stability, sub-cellular local-zation, and can have a different target spectrum.

This study introduces a data analysis pipeline to filter, annotate,nd detect miRNAs and their different isomiRs. The workflow isainly based on standard Galaxy tools and a number of in-house

cripts. Well-annotated related species information was used tomprove the annotation of each sequence found in small RNA-Seqesults. The pipeline consists of basic analysis steps to check foruality, filtering, and clipping the adapter sequence. Afterwards allequences were collapsed to unique sequences and the correspond-ng read counts. These reads were mapped with BLASTn-shorto align them to all transcripts of our sequenced species, includ-ng ncRNAs and related well-annotated species. The collection ofLAST databases contained sequences from mirBase (precursornd mature miRNAs), sequences from NCBI and Ensembl, includ-ng ncRNAs and protein-coding transcripts, as well as tRNAs andiRNA cluster sequences. All BLAST results were filtered and joinedy removing duplicated hits, which resulted in ∼45,000 mappedequences out of ∼68,000 unique filtered sequences. The annotatedequences were further analyzed for isomiR differences. To do that,′ end and 5′ end modifications were detected and analyzed by type.inally, expression levels were compared within isomiR groups andowards the corresponding mature miRNA. Using this annotationipeline, 1581 unique sequences could be assigned to 266 differentiRNAs. The highest expression levels were found for the canoni-

al miRNA form (mature miRNA) (∼70%) within the miRNA groups
ncluding the mature miRNA (172 group members), followed bysomiRs with a 3′ end modifications with (∼23%). In groups withsomiRs without the presents of the canonical miRNA (94 group
embers), the 3′ end modified isomiRs had the highest expres-


sion levels with 74%, and 21% at 5′ end followed by 5% on bothsides.


PO 50Development of miRNA profiling capacity usingreverse transcription-quantitative PCR(RT-qPCR) by Prestizia, a miRNA platform forbiomarkers signature discovery

Marjorie Monleau, Gilles Vieira ∗,Laurent Tosolini, Odile Prigneau

Prestizia, FranceE-mail address: [email protected]

Context and objectives: The discovery of microRNAs (miRNA)in body fluids paves the way for the characterization of novel non-invasive biomarkers. The relative abundance of those miRNA can bequantified thanks to several platforms and technologies. However,the final validation of their expression uses the RT-qPCR method.The biomarker discovery nowadays is based on high-throughputpipelines, and for miRNA, a commercial solution is available withthe Life Technologies TaqMan Array Human MicroRNA cards A andB. Although the two cards provide 760 miRNA, more than 2000are now described in the literature and specific databases. Our aimwas to increase the capacity of miRNA profiling by designing newcustom cards. In parallel we developed a standardized workflow toanalyze simultaneously the data provided by both the commercialand custom cards.

Methodology: The design of the new custom cards was achievedby using the miRBase information. To validate the newly designedcards, we used the same set of standardized positive control(human tissues RNA and human serum pool) and negative control.This allowed us to determine and assess the detection capacity,reproducibility, sensitivity, differential expressions and specificity.Finally we tested matched serum and whole blood samples fromdifferent donors.

Results: The detection capacity was first tested on a largenumber of new miRNAs (4 custom cards with a total of 1440 miR-NAs): 516 miRNAs were detected and were subsequently used todesign a second version of two custom cards “C and D”. Analyti-cal performances of these two custom cards were compared to thecommercially available A and B cards.

The aim of miRNA expression profiling studies is to quantifydifferences between sample groups; we showed that we were ableto quantify differentially expressed miRNAs between two sampletypes.

Conclusion: Finally, we have successfully validated 2 new cards

dx.doi.org/10.1016/j.bdq.2017.02.137



dx.doi.org/10.1016/j.bdq.2017.02.138



dx.doi.org/10.1016/j.bdq.2017.02.139

lar Det

PTa

PR

1

2

E

ilttotpccphmodipbRTa

h

PTp

HJCJ

1

R2

(Eh

aatptcibipCtrdE


O 51wo-tailed RT-qPCR: A novel method for highlyccurate MiRNA quantification

eter Androvic 1,∗, Lukas Valihrach 1, Julie Elling 2,obert Sjoback 2, Mikael Kubista 1,2

Institute of Biotechnology CAS, CzechiaTATAA Biocenter AB, Sweden-mail address: [email protected]

MicroRNAs are a class of small non-coding RNAs that serve asmportant regulators of gene expression at the posttranscriptionalevel. MiRNAs are stable in body fluids and pose great potentialo serve as biomarkers. Here, we present a highly specific, sensi-ive and cost-effective system to quantify miRNA expression basedn two-step RT-qPCR with SYBR-green detection chemistry calledwo-tailed RT-qPCR. It takes advantage of novel, target-specificrimers for reverse transcription composed of two hemiprobesomplementary to two different parts of the targeted miRNA,onnected by a hairpin structure. The introduction of a secondrobe ensures high sensitivity and enables discrimination of highlyomologous miRNAs irrespectively of the position of the mis-atched nucleotide. Two-tailed RT-qPCR has a dynamic range

f 8 logs and a sensitivity sufficient to detect down to a hun-red of target miRNA molecules. It is capable to capture the full

somiR repertoire, leading to accurate representation of the com-lete miRNA content in a sample. The reverse transcription step cane multiplexed and the miRNA profiles measured with Two-tailedT-qPCR show excellent correlation with the industry standardaqMan miRNA assays (R2 = 0.985). Moreover, two-tailed RT-qPCRllows for rapid testing with a total analysis time of less than 2.5 h.


O 52etraspanin CD9 modifies the RNA cargo ofrostate extracellular vesicles

elen Jankowski 1,∗, Belinda Goldie 1,2,oshua Brzozowski 1, Benjamin Munro 1,hristopher Scarlett 1, Kathryn Skelding 1,

udith Weidenhofer 1

University of Newcastle and Hunter Medicaleasearch Institute (HMRI), AustraliaInstitute for Integrated Cell-Material Sciences

iCeMS), Kyoto University, Kyoto, Japan-mail address:[email protected]

Introduction: Extracellular vesicles (EVs) have gained interests a promising avenue for cancer biomarkers in recent years. EVsre small spherical shaped vesicles, which are secreted from theirissue of origin and contain high amounts of noncoding RNAs. Therocess for RNA loading into EVs is still not fully understood, buthere are likely to be multiple mechanisms potentially based onell type and disease. Potential components that may play a rolen cargo sequestering are tetraspanins. They have been shown toe involved in key cancer-related cellular processes, with stud-

es showing that expression of the tetraspanin CD9 correlate withrostate tumour type, stage and patient outcome. A decrease inD9 expression is indicative of poor prognosis and metastasis. Fur-
her, CD9 is abundant on EVs that represent a source of biomarkerseadily accessible by non-invasive means. This project seeks toetermine how CD9 expression in prostate cancer influences theV cargo to identify novel prognostic biomarkers.

Methods: CD9 expression was knocked down in RWPE1 (normalprostate cells) and increased in PC3 (bone metastasis from prostatecancer). EVs were collected after 48 h by ultraconcentration fromsupplement-free cell culture media. NanoSight NS300 was usedto evaluate size and concentration by nanoparticle tracking analy-sis. Total RNA was extracted with Trizol and evaluated using totalRNA Agilent 2100 bioanalyzer chips, RNA was converted to labeledcDNA and hybridized to Affymetrix Human Transcriptome arrays,gene level differential expression (p < 0.05) and alternative splicinganalyses were performed (event score ≥0.7).

Results: Increasing the expression of CD9 in PC3 cells resultedin increased incorporation of CD9 in EVs and the differential incor-poration of 2532 transcripts. Whereas decreasing CD9 expressionin RWPE1 cells and the resultant EVs altered the incorporation of486 transcripts into the EVs. A highly incorporated transcript fam-ily, particularly in the CD9 knockdown cells, is the small nucleolarRNAs, whose function is to chemically modify RNA. The alternativesplicing analysis suggests that increased CD9 expression in prostatecancer cells and EVs results in incorporation of 10 alternativelyspliced transcripts in EVs, whereas decreased CD9 expression innormal prostate cells generates EVs with 7 unique splicing events.What is not known, as this stage is if these transcripts are full lengthor are fragments.

Conclusion: These transcripts therefore represent novel poten-tial EV biomarkers due to being differentially incorporated byexpression of the metastasis suppressor CD9. From this it may bepossible to identify a biomarker signature that will improve out-comes for prostate cancer patients.


PO 53EXÖBead: An efficient method of isolatingexosomes from small sample volumes withoutultracentrifugation

Dapi Chiang 1,∗, Dominik Buschmann 2,Benedikt Kirchner 2, Michael W. Pfaffl 2

1 Biovesicle Inc., Taiwan2 Division of Animal Physiology and Immunology,School of Life Sciences Weihenstephan, TechnicalUniversity Munich, GermanyE-mail address: dapi [email protected]

Exosomes are small vesicles (30–150 nm) secreted from differ-ent cell types and found in various biofluids, such as blood, urine,saliva and CSF. Exosomes contribute to cell–cell communication,antigen presentation or tumor progression by carrying cellularproteins, RNA/DNA, glycans and lipids. Differential ultracentrifu-gation is still regarded the ‘Gold Standard’ for isolation exosomes.However, ultracentrifugation is a laborious and time-consumingmethod that requires specialized equipment and operationalexpertise. Several alternative methods such as polyethylene glycol(PEG) or antibody-conjugated beads were developed to isolate exo-somes without ultracentrifugation. These methods reveal differentabilities of exosome RNA or protein isolation. A major issue for PEGisolation is the purity of exosome due to PEG remains. Isolationbased on antibody-conjugated beads, however may damage exo-somes by using acidic or alkaline reagent to break antigen-antibodyinteractions. To solve these issues, we create a non-antibody coatedmagnetic bead, EXÖBead that is able to isolate exosomes in a
very elegant and efficient way without ultracentrifugation. Weincubated EXÖBead with minimal volumes of pre-cleared cell cul-ture medium or serum and analyzed the pulled-down fraction byflow cytometry (FACS), western blot, bioanalyzer and nanoparticle


dx.doi.org/10.1016/j.bdq.2017.02.140



dx.doi.org/10.1016/j.bdq.2017.02.141



S lar Det

tmp2suawEE(btbdtftis

h

PDda

A

Ea

tortMafaaaoccdicmsgadRaafgaaeP

ity of clinical oncology specimens collected in cfDNA BCTs with theBEAMing technology.



racking analysis (NTA). Our result showed that exosome proteinarker CD63 can be detected by FACS in exosome-EXÖBead com-

lexes from 100 �l to 1 ml human serum and 200 �l to 1 ml RAW64.7 cells culture medium. Additionally, the expression of exo-omal proteins Alix and Rab5 was substantiated by western blotsing the exosome-EXÖBead complex from 200 �l mouse serumnd 200 �l to 1 ml B16F10 cells culture medium. For RNA analysis,e used a commercial extraction kit to extract RNA from exosome-

XÖBead complex. The pattern of exosomal RNA and its cDNA fromXÖBead isolation is similar with differential ultracentrifugation120,000 × g pellet). Furthermore, we designed a specific elutionuffer for EXÖBead without using any acidic or alkaline reagent. Toest the ability of EXÖBead elution buffer to release exosomes fromeads, we performed NTA analysis to measure exosome size and itsistribution. NTA data showed the major peaks of eluted exosomeso be at 65 nm, 105 nm and 155 nm. The size of exosomes elutedrom EXÖBead is similar to exosomes isolated by ultracentrifuga-ion. In conclusion, EXÖBead provide an easy, fast and reproduciblesolation method for researcher working with small amounts oftarting material.


O 54roplet digital PCR: Analysis approach for theetection and absolute quantification of lacticcid bacteria

ntje-Kristin Mommsen ∗, Stephan Scholtz

DuPont Danisco Deutschland GmbH, Germany-mail address:[email protected]

This study demonstrates the application of the droplet digi-al PCR (ddPCR) method, namely the absolute cell quantificationf lactic acid bacteria in multi species cultures. Lactic acid bacte-ia comprise a wide range of species that produce lactic acid ashe major metabolic end product of carbohydrate fermentation.

ulti species lactic acid bacterial starter cultures are tradition-lly used for the production of a variety of cheeses and freshermented milk products. These starter cultures are indispens-ble for complex fermentation processes that not only require thecidification of the milk but also to produce the desired flavornd to extend the shelf life of the product. Standard microbi-logical analyses based on cultivation on selective agar mediaan identify lactic acid bacteria but they are laborious and time-onsuming. Additionally, they clearly have some limitations iniscriminating species showing similar physiological character-

stics or to quantify particular species in complex cultures thatan comprise different lactic acid bacteria species. Therefore, aethod was supposed to be established that can quantify the

pecies of interest in a mix of species independently from the back-round cell concentration in order to replace the time-consumingnd laborious culturing techniques. For this purpose the dropletigital PCR method was applied using the instrument QX200 (Bio-ad). A comparison study between plate count analyses of lacticcid bacteria multi species cultures and ddPCR revealed that thebsolute cell counts were by trend higher using ddPCR. There-ore, the absolute quantification of cells is expressed as absoluteenomic units (AGU). It could be shown that the inter-run vari-bility of ddPCR was low with 2–8%. In addition, a fiveplex ddPCR
ssay was set up in order to quantify simultaneously five differ-nt species in a multi species starter culture. Using this fiveplexCR, the absolute number of AGU’s of different species common in

several product samples can be quantified within a single ddPCRreaction.


PO 55Clinical samples collected in Streck Cell-FreeDNA blood collection tubes are suitable forliquid biopsy testing using BEAMing digital PCR

Annette Nocon 1,∗, Inga Medina Diaz 1,Makbule Kobilay 2, Dirk Skowasch 2,Stefanie Held 2, Claudia Stamm 1, Frank Diehl 1,Stefan Holdenrieder 2, Frank Holtrup 1

1 Sysmex Inostics GmbH, Germany2 University Hospital Bonn, GermanyE-mail address:[email protected]

Mutation profiling of circulating cell-free DNA (cfDNA)extracted from plasma samples has advanced to a powerful diag-nostic tool in oncology. Making liquid biopsy tests broadly andeasily available requires the transport of whole blood to a centraltesting laboratory. In standard K2EDTA tubes, white blood cells startto lyse shortly after blood collection, thereby releasing genomicDNA into the plasma and diluting the already small amounts ofcirculating tumor DNA (ctDNA). Streck Cell-Free DNA blood col-lection tubes (cfDNA BCTs) were specifically designed to maintainblood cell integrity and cfDNA stability for several days. Thus, cfDNABCTs present a promising alternative to standard K2EDTA tubes andhave been integrated in many liquid biopsy workflows.

However, qualification data for the use of Streck cfDNA BCTsin oncology is limited and mainly based on blood collected fromhealthy individuals as well as data extrapolated from the prena-tal testing field. The data generated from these samples may notrepresent the unique dynamics of clinical oncology specimens andtherefore a study of true clinical oncology samples is required tosupport the use of cfDNA BCTs in routine practice.

In this study, we evaluated the cfDNA integrity of matchedblood samples collected in cfDNA BCTs vs standard K2EDTA tubesfrom colorectal, pancreatic and non-small cell lung cancer patients(N = 53). Blood drawn into cfDNA BCTs was either processedimmediately or 3 days after phlebotomy. DNA quantification wasfollowed by BEAMing digital PCR (OncoBEAMTM) on KRAS, NRASand EGFR mutations and compared to matching specimens col-lected in K2EDTA tubes.

Our results suggest that cfDNA yield as well as the genomicDNA background is not affected by prolonged storage of clinicalsamples in cfDNA BCTs for up to 3 days. In all sample sets contain-ing mutant ctDNA, the detected mutational load was comparablebetween cfDNA BCTs and K2EDTA tubes.

In conclusion, this study represents a comprehensive clinicalevaluation of cfDNA BCTs vs EDTA tubes for ctDNA profiling. Inconjunction with the findings of our previously presented cfDNABCT shipping condition studies, our data supports the compatibil-

dx.doi.org/10.1016/j.bdq.2017.02.142



dx.doi.org/10.1016/j.bdq.2017.02.143



dx.doi.org/10.1016/j.bdq.2017.02.144

lar Det

PDmn

PRR

1

VA2

VE1

rdDiot7cbq

cnt–ccS

IR

[

[

h

PPQ

NT

En

noevscees

the TMPRSS2:ERG fusion as a suitable biomarker.For Research Use Only. Not for use in diagnostic procedures.



O 56igital PCR quantification of the 7S DNA peritochondrial genome proportion in

on-invasive sources of human cell types

hilipp Steindorf 1,∗, Martin Hofer 1,oman Ziegler 1, Bernard Wallner 2,alf Steinborn 1

Genomics Core Facility, VetCore, University ofeterinary Medicine, Veterinärplatz 1, Vienna,ustriaDepartment of Behavioural Biology, University ofienna, Vienna, Austria-mail address:[email protected]

The mitochondrial displacement (D-) loop is a triple-strandedegion found in the major non-coding region of many mitochon-rial genomes. It is formed by stable incorporation of a thirdNA strand known as 7S DNA [1]. This extra strand – approx-

mately 650 nucleotides in length – starts around the originf heavy strand replication (OH) and terminates just after theermination-associated sequence. The few reported proportions ofS DNA-containing molecules ranging from 10% in cultured humanells to 90% of Xenopus oocytes (reviewed in [1]) have exclusivelyeen performed by semi-quantitative methods before the era ofuantitative PCR (qPCR) or digital PCR (dPCR).

Here we determined the proportion of 7S DNA per mito-hondrial genome in a number of human cell types sampledon-invasively from healthy individuals. Copy number concen-rations of target nucleic acids were determined by digital PCR

the gold standard for their precise quantification – using thehip-based platform QuantStudioTM 3D Digital PCR System inombination with the QuantStudioTM 3D AnalysisSuiteTM Cloudoftware (Thermo Fisher Scientific) [2].

This work is supported by the University of Vienna (projectP547012 to B.W.).eferences

1] T.J. Nicholls, M. Minczuk, In D-loop: 40 years of mitochondrial 7S DNA, Exp.Gerontol. 56 (2014) 175–181.

2] F.R. Belmonte, et al., Digital PCR methods improve detection sensitivity andmeasurement precision of low abundance mtDNA deletions, Sci. Rep. 6 (2016)25186.


O 57oisson plus quantification for theuantStudioTM 3D Digital PCR System

ivedita Majumdar ∗, Swapnonil Banerjee,homas Wessel, Mike Pallas, Patricia Hegerich

Thermofisher Scientific, United States-mail address:[email protected]

Digital PCR, a next generation nucleic acid quantification tech-ique works by spreading the target material across a large numberf partitions. The average number of molecules per partition isstimated using Poisson statistics, and this partition average is con-erted into concentration by dividing by partition volume. In thistandard approach, identical partition sizing is assumed. At higher
oncentrations, violations of this assumption result in the under-stimation of target quantity, when using Poisson modeling. Anxtension to the Poisson model, the Poisson-Plus Model, is pre-ented here; which, if statistics of the volume variation are well

characterized, accommodates for this underestimation. Parametersof the model were measured on the chip array based QuantStu-dio 3D Digital PCR System. The volume variation was measuredusing the ROX fluorescence signal level as a proxy for effective loadvolume per through-hole. Monte Carlo simulations demonstratethe efficacy of the proposed correction. Empirical measurementof model parameters characterizing the effective load volume onQuantStudio 3D Digital PCR chips is presented. The model wasthen used to analyze digital PCR experiments from QuantStudio3D Digital PCR System and showed improved accuracy in quantifi-cation results. At the higher end of the concentration spectrum, themodeling must take effective volume variation into account to pro-duce the most accurate estimate. The extent of the difference fromthe standard to the new modeling is proportional to the extent ofvolume variation in the effective load of your reactions.


PO 58Detection of TMPRSS2:ERG fusion transcriptusing TaqMan assays and the QuantStudio 3DDigital PCR System

Alice Rödel 1,2,∗, Marion Laig 3, Arndt Schmitz 1

1 Bayer AG, Berlin, Germany2 Beuth University of Applied Sciences, Berlin,Germany3 Thermo Fisher Scientific, South San Francisco, CA,United StatesE-mail address: alice [email protected]

Current biomedical research aims at personalized cancer thera-peutics, for example in prostate cancer. Due to the high prevalenceof the TMPRSS2:ERG fusion which occurs in more than 50% of cases,it seems to be a suitable biomarker for monitoring prostate can-cer which can be detected in less invasive sample material suchas blood and urine. Use of digital PCR is currently being demon-strated to be a highly sensitive method for reproducible and robustmeasurements without the use of standard curves. Especially theQuantStudio 3D Digital PCR System from Applied Biosystems hasa high point-of-care potential due to the closed, compact and easyto handle system. Aim of this project was to establish a system forthe detection of TMPRSS2:ERG fusion on this Digital PCR platform,which can be deployed as a future-oriented method. A TaqManFusion and a Gene Expression Assay were used to detect mutant andwildtype alleles, and their performance was optimized by chang-ing the temperature-time profile. RNA isolated from fusion positiveVCaP and fusion negative LNCaP cell lines were used as refer-ence material. In wet-lab experiments it was possible to detectTMPRSS2:ERG transcripts with a detection limit of 0.05% fusionportion in a correspondingly high wildtype background. Spike-inexperiments allowed the confirmation of an easy workflow. This isa first step forward to apply the assay to prostate cancer samplesfor monitoring disease and to further validate the significance of



dx.doi.org/10.1016/j.bdq.2017.02.145



dx.doi.org/10.1016/j.bdq.2017.02.146



dx.doi.org/10.1016/j.bdq.2017.02.147

S lar Det

PTSum

JM

E

eaolftm

cbblaspbc

Fflienstfdinf

mSlc

h

PDt

NM

En

agos


O 59oward elucidation of the biological function of100 calcium binding protein A9 in monocytessing single cell sorting and qPCRethodologies

ianxin Yang ∗, Carin van der Keur, Frans Claas,ichael Eikmans

LUMC, The Netherlands-mail address: [email protected]

Background: We previously found that relatively high tissuexpression of S100 calcium binding protein A9 (S100A9) earlyfter clinical kidney transplantation is associated with favorableutcome at the long run. S100A9 is abundantly expressed by circu-ating monocytes and tissue macrophages. To clarify the biologicalunction of S100A9, we now aim to both functionally and pheno-ypically characterize S100A9 high expressing and -low expressing

onocyte subsets.Method: We addressed our aim by two approaches: (I) appli-

ation of SmartFlareTM (Merck Millipore) RNA detection probesound to nanoparticles. These should be taken up within 16 hy live monocytes, and fluoresce once they bind intracellu-

ar S100A9 mRNA targets; (II) performing intracellular stainingnd sorting for S100A9 protein by flow cytometry (FACS), andubsequent qPCR profiling of the cell fractions. For both pur-oses, CD14+ human monocytes were employed, which hadeen enriched from Ficoll-isolated mononuclear cells of buffyoats.

Results: Monocytes incubated with S100A9-specific Smart-lares were gated on high, intermediate, and low signals byow cytometry. The three different fractions did not differ in

ntracellular S100A9 protein, and also not in S100A9 mRNAxpression levels after FACS sorting. Therefore, cell fractions wereot further investigated in functional tests. Intracellular proteintaining normally disrupts the RNA and poses a challenge inhe reliable molecular profiling of cellular mRNA content. Weound that a specialized buffer containing 5% RNAse inhibitoruring cell sorting kept mRNA degradation to a minimum (Cq

ncrease < 3). Most importantly, S100A9high fractions expressed sig-ificantly higher (∼two-fold) S100A9 mRNA levels than S100A9low

ractions.Conclusion: The SmartFlare technology does not generate

onocyte fractions that differently express S100A9. Intracellular100A9 protein staining of monocytes enables sorting of high- andow-expressing cell subsets. This will enable further phenotypicalharacterization of the subsets.


O 60etermining the limit of detection of rare

argets using Digital PCR

ivedita Majumdar ∗, Thomas Wessel,arion Laig, Brian Ho, David Keys

Thermofisher Scientific, United States-mail address:[email protected]

Detection and quantification of mutant alleles in tumor tissue
llow for disease monitoring, the evaluation of drug efficacy anduide decisions on future treatment plans. Testing for the presencef mutations in circulating free DNA (cfDNA) is one of the less inva-ive research methods available at this time. Digital PCR presents a

research tool for mutation detection in cfDNA at a sensitivity levelof 1% and below.

Challenges associated with digital PCR experiments for rareallele detection include understanding the limit of detection ofthe assay and platform. This work compares false positive assess-ment strategies using the signal levels of the no-amplificationcluster. Once the false positive call rate is established, this workoutlines a method to determine the limit of detection of theassay and platform, at a given level of confidence. Given thenumber of partitions, the interrogated volume and the false callrate, the tradeoffs between sample load and sensitivity are alsodiscussed.

The mathematics outlined to calculate the theoretical limit ofdetection is applied on a set of assays from Thermo Fisher Scien-tific covering the KRAS codon mutations commonly found in tumortissues. Experimental results showing a detection of at least 0.1%mutation rate are presented as examples. Test samples were cre-ated using both mutant plasmid and mutant genomic DNA mixedwith wild-type genomic DNA at a predefined percentage.

For Research Use Only.


PO 61Single cell gene expression profiling in breastcancer cells with the Her2/neu gene knockoutby CRISPR-Cas9

Xiaoyang {Alice} Wang 1, Chip Lomas 1,Michael A. Tycon 1, Craig Betts 2,Wieland Keilholz 2,∗, Suzanne Weaver 2

1 BD Biosciences2 BD GenomicsE-mail address: [email protected]

The Her2/neu gene is amplified and overexpressed in 15–30%of breast cancers. The overexpression of this oncogene is stronglycorrelated with decreased survival, increased cancer relapse, andpoor prognosis. Although therapy has been successfully devel-oped to target this oncogene, a better understanding of thisoncogene will provide further insight for breast cancer biologyand future drug development. In recent years, the CRISPR-Cas9system has emerged as an efficient method for genetic engi-neering, enabling targeted gene knockout with minimal off-targeteffects.

In this study, we employed a plasmid-based CRISPR-Cas9 systemto knock out the Her2/neu gene in breast cancer cells. Puromycinselected, GFP-positive (two indicators of the incorporation of theCRISPR-Cas9 system), Her2-negative individual cells were index-sorted using a BD FACSMelodyTM system into individual wells ofa 96-well BDTM Precise plate with sample barcoding and molec-ular indexing. A whole transcriptome amplification (WTA) assaywas performed to obtain a gene expression profile for each ofthe Her2/neu knockout cells. Her2 gene knockout was performedon two cell lines: T47D, a ductal carcinoma-derived breast cancercell line with no Her2 amplification that shows low to intermedi-ate Her2/neu expression; and SKBR3, an adenocarcinoma-derivedbreast cancer cell line known for Her2/neu gene amplification andoverexpression. For comparison, WTA assays were also carried outon parental cells without gene editing. Comparison of the geneexpression profiles reveals downstream changes in gene expres-
sion correlated to the Her2 knockout.
The profiling of gene expression in Her2/neu CRISPR-Cas9knockout cells on a single cell level promises to provide insight



dx.doi.org/10.1016/j.bdq.2017.02.148



dx.doi.org/10.1016/j.bdq.2017.02.149



lar Det

if

h

PTmh

CJW

E

ptttnfomeaatioihfcstwiettdigs

h

PHarp

KGC

E

uic


nto the mechanisms in these aggressive cancers that could helputure drug discovery.


O 62cell differentiation dynamics profiled byassively parallel single cell RNA capture and

ighly multiplexed PCR

hristina Chang, Eleen Shum, Nidhanjali Bansal,oanna Dreux, Gretchen Lam, Jue Fan,

ieland Keilholz ∗, Christina Fan

BD Genomics-mail address: [email protected]

The immune system consists of a complex network of cellopulations that can go through different spatial and temporalransitions. Cell surface markers and flow cytometry have aidedhe characterization of immune subsets. However, restrictions inhe number of available antibodies and multicolor cytometry chan-els greatly limit access to phenotypic information that illuminate

unction and cell identity. Single cell whole transcriptome analysisffers a higher resolution approach to uncover novel regulators andarkers obscured by bulk sample analysis. Yet several limitations

xist for such approach for T cells. T cells, especially in an un-ctivated state, have low numbers of transcripts for surface markersnd transcription factors used to define lineage and function, andhus deeper sequencing is required to accurately detect genes ofnterest. Moreover, a great proportion of sequencing reads consistf ribosomal and housekeeping genes, making meaningful cluster-ng based on cell identity difficult. Here we demonstrate using aighly multiplexed PCR approach to characterize T cell lineage and

unction with greater sensitivity for T cell specific genes and higherost effectiveness. The BDTM Resolve targeted T cell assay allows theurveying of more than 300 T cell genes in hundreds to more thanen thousand of individual cells per experiment. In this study weere able to distinguish between T cells that were differentiated

nto different CD4 effector subsets using cluster analysis. Withinach condition, we were able to identify cells in different activa-ion and differentiation states. With much fewer sequencing readshan what is needed for the whole transcriptome approach, weetected low abundance T cell markers that are hard to uncover

n whole transcriptome sequencing. The BD Resolve single cell tar-eted approach is a high throughput method to study the functionaltates of T cells in a high dimensional and cost effective way.


O 63igh throughput single-cell gene expressionnalysis reveal cell type specific differences inesponse to different human peripheral bloodreservation protocols

imberly R. Cordes Metzler, Nidhanjali Bansal,retchen Lam, Jue Fan, Wieland Keilholz ∗,hristina Fan

BD Genomics-mail address: [email protected]

Gene expression analysis of peripheral blood has been widelysed to identify biomarkers for detecting, monitoring, and predict-

ng diseases. However, it has been known that gene expressionhanges in blood can be induced by different blood handling and


processing methods, thus skewing experimental results. Past stud-ies examining effects of blood handling and preservation havemostly been conducted with bulk RNA using microarrays, and itis not commonly known whether certain cell populations in bloodare more prone to gene expression changes. Here, we used BDResolve, a tool for massively parallel single-cell RNA sequencinglibrary preparation, and shallow sequencing, to compare wholetranscriptome profiles of ∼10,000 freshly isolated peripheral bloodmononuclear cells (PBMCs) each from three blood donors, as wellas blood cells isolated and/or stored in the following conditions:freshly isolated, cryopreserved, or liquid life AQIX

®storage. With

such high resolution single cell analysis, we found donor to donordifferences, not only in population sizes of immune subsets, butalso in gene expression differences in each of the subset. Addition-ally, we found that certain immune subset, such as monocytes, weremore susceptible to expression changes depending on preservationtechniques. Together these results will help develop proper meth-ods for handling and preserving blood samples for future large scalehigh throughput single cell gene expression studies.


PO 64Preamplification using dUTP and Cod UNGallows for reliable detection and quantificationof limited samples sizes in the presence ofcontaminating DNA amplicons

Daniel Andersson 1,∗, David Svec 1,2,Cathrine Pedersen 3, Jørn Remi Henriksen 3,Anders Ståhlberg 1

1 Sahlgrenska Cancer Center, Department ofPathology, Sahlgrenska Academy at University ofGothenburg, Box 425, 40530 Gothenburg, Sweden2 Institute of Biotechnology, Academy of Sciences ofthe Czech Republic, Videnska 1083, Prague 4, 14221,Czechia3 ArcticZymes AS, Sykehusveien 23, 9019 Tromsø,NorwayE-mail address: [email protected]

Detection and quantification of rare DNA and RNA moleculesin limited sample sizes, including liquid biopsies, fine-needleaspirates and single cells, usually requires preamplification. Thismakes downstream analyses especially sensitive to PCR generatedcontamination. Here, we study the feasibility to perform preampli-fication with dUTP in the presence of Cod uracil-DNA N-glycosylase(Cod UNG), allowing elimination of contaminating DNA amplicons.Cod UNG can be completely and irreversibly heat inactivated, mak-ing it suitable for preamplification applications. Using optimizedqPCR assays, we show that using dUTP instead of dTTP in the reac-tion mix yields similar dynamic range, reproducibility, sensitivityand amplification efficiency. Furthermore, we demonstrate thatCod UNG degrades essentially all contaminating uracil-containingamplicons regardless of initial concentration in all samples for mostassays tested without inhibiting downstream reactions. Finally, wevalidate that dUTP and Cod UNG can easily be included in theworkflow for targeted single-cell gene expression profiling. In con-clusion, a standardized introduction of dUTP and Cod UNG in theexperimental workflow poses an attractive solution to largely avoidgeneration of erroneous data when analyzing limited sample sizes
containing few target molecules, in clinical routine assessments aswell in research studies.

dx.doi.org/10.1016/j.bdq.2017.02.150



dx.doi.org/10.1016/j.bdq.2017.02.151



dx.doi.org/10.1016/j.bdq.2017.02.152



dx.doi.org/10.1016/j.bdq.2017.02.153

S lar Det

PAr

SCM

1

2

Es

ctlbaampDaLstlrct

h

PRido

K

TE

alasmeeshcw“dpcthvft


O 65chieving unparalleled sensitivity andeproducibility in single-cell transcriptomics

ara Gonzalez-Hilarion 1,∗, Kazuo Tori 2,ynthia Chang 2, Yevgeniy Gindin 2,agnolia Bostick 2, Andrew Farmer 2

Takara Bio Europe, FranceTakara Bio USA-mail address:ara [email protected]

Differential expression (DE) analysis utilized for single-cellomparisons has become one of the key methods for studyingranscriptome variability, especially when homogeneous cell popu-ations are elusive, such as in cancer research, developmentaliology, neurobiology, and immunology. SMART (Switching Mech-nism at the 5′ end of the RNA Template) technology has emergeds the most sensitive solution for processing the small amounts ofRNA present in a single cell. Here we discuss the use of the highly

erformant SMART-Seq v4 chemistry in the new SMART-Seq v4 3′

E Kit to enable differential expression analysis in a more efficientnd cost-effective manner. SMART-Seq v4 chemistry incorporatesNA technology in order to produce high-quality, reproducibleequencing data with superior identification of genes, includinghose with low expression. By combining these features with cel-ular indexes, pooling, and 3′ end-capture sequencing, we enableesearchers to obtain high-quality gene expression data from singleells or ultra-low input amounts of RNA without having to sequencehe entire transcriptome.


O 66esolving molecular networks and dynamics

nvolved in CD8+ T-cells function andifferentiation in acute and chronic infectionsn a single-cell level

ristiyan Kanev ∗, Patrick Roelli, Dietmar Zehn

Division of Animal Physiology and Immunology,echnical University of Munich, Germany-mail address: [email protected]

CD8 T-cells are major players in the adaptive immune defencegainst intracellular pathogens (including viruses and intracellu-ar bacteria) as well as in tumor surveillance. In the context ofcute viral infections like influenza and yellow fever, naive antigen-pecific CD8 T-cells differentiate into highly functional effector andemory CD8 T-cells. In result, the generated effector cells swiftly

liminate the viral infections, while the memory cells provideffective protection in case of secondary infection caused by theame virus. In contrast, some viruses like HIV, HBV and HCV inumans as well as LCMV clone 13 in mice are able to establishhronic infections associated with the development of CD8+ T-cellsith diminished functional activity, a state often referred as T-cell

exhaustion”. The latter represents a unique state of CD8 T-cellifferentiation which is likely to be effective but hyporesponsive,roviding the host with the ability to control the viral load withoutausing severe bystander pathology. Otherwise the excess cyto-oxic T-cell activity due to persisting antigen presence can bring the
ost to even more life-threatening condition compared to chroniciral presence. Some of the general CD8 T-cell hallmarks in dif-erent chronic infections compared to acute infection are uniqueranscriptional program, immune signaling, migration as well as

metabolism. Despite the amount of accumulated knowledge aboutCD8 T-cell phenotype and function in chronic infection, we arestill far away from establishing successful strategies for therapeuticreactivation of the immune system in order to eradicate establishedchronic infections. In our opinion, this might be a reflection of theusually applied population-based assessment of CD8 T-cell geneexpression profiles, which is prone to generating biased and evenfalse results due to giving only average values. The main questionwe want to address is which molecular networks and mechanismare involved CD8 T-cell function and differentiation in acute andchronic infections on a single cell level. To address that questionwe decided to generate gene expression profiles of CD8 T-cellsfrom different acute and chronic infection settings and time pointsusing single-cell mRNA sequencing approach. So far we are opti-mizing the method and we have started to obtain single-cell geneexpression profiles from different settings. Besides the deep single-cell information on molecular networks and mechanism, we hopethat the single-cell approach we apply will be informative concern-ing cellular dynamics and subpopulation taking place in differentacute and chronic infections. Finally, the ultimate outcome will beto exploit that knowledge for generating new therapeutic strategiesfor successful intervention in immune response against problem-atic viral infections.


PO 67Combining microgenomics methods to studyhow gut microbiota influences gene expressionin the hypothalamo-pituitary-adrenal axis inrats

Patricia Anglade 1, Elise Maximin 1,Nicolas Crapart 2,3, Jad Abou-Ghantous 4,Marie-Noelle Rossignol 2, Philippe Gerard 1,Patrice Martin 2, Claudia Bevilacqua 2,Sylvie Rabot 1,∗, Bénédicte Goustard 1

1 Micalis Institute, INRA, AgroParisTech, UniversitéParis-Saclay, Jouy-en-Josas, France2 GABI-Plareforme @BRIDGe, INRA, AgroParisTech,Université Paris-Saclay, Jouy-en-Josas, France3 EXCILONE, Elancourt 78990, France4 MGP MetaGenoPolis, INRA, Université Paris-Saclay,Jouy-en-Josas, FranceE-mail address: [email protected]

Objective: The gastro-intestinal tract hosts a complex micro-bial community, the gut microbiota, which is nowadays regardedas a full organ taking part to the host physiology. The gut micro-biota is in particular increasingly recognized as a factor thatcan shape brain and behaviour. We and others showed that thegut microbiota regulates the hypothalamo-pituitary-adrenal (HPA)axis reactivity, as reflected by a greater corticosterone systemicconcentration in germfree rodents than in conventional ones, fol-lowing an acute stress. The objective of the present study was todevelop a microgenomics workflow combining the use of lasercapture microdissection (LCM), pre-amplification and reverse tran-scription real time PCR (RT-qPCR) to analyse which HPA axis genesare regulated by the gut microbiota.

Methodology: We subjected germfree and conventional rats toan acute stress (strongly illuminated open-field for 6min), killed
them by decapitation and collected and froze the brain, the pituitarygland and the adrenal glands. Non stressed rats served as controls.Different combinations of analytical methods were used in those3 tissues, according to the distribution and density of the cells of


dx.doi.org/10.1016/j.bdq.2017.02.154



dx.doi.org/10.1016/j.bdq.2017.02.155



lar Det

ivtiatapcwCaTst

Micro Fluidic Cards. We acknowledge Excilone for supporting LCM


nterest. Brain and adrenal gland sections were stained with cresyliolet to localize the key areas, namely the hypothalamus paraven-ricular nucleus (PVN) in the brain, and the cortical zona fasciculatan the adrenal glands. Then, distinct cell harvesting techniques werepplied: micro-punching for the PVN, and LCM of cell clusters forhe adrenal gland. As the corticotropic cells are scattered in thenterior pituitary gland, an immunohistochemistry method cou-led with LCM was developed to identify and capture corticotropicells as specifically as possible. Around 50 corticotropic single cellsere isolated and processed with the TaqMan

®PreAmp Cells-to-

TTM kit. The expression level of a panel of 48 genes, selected

ccording to their role in the HPA axis reactivity, was analysed usingaqMan

®Array Micro Fluidic Cards; qPCR was carried out in all

orted samples. A target-specific pre-amplification was applied inhe adrenal gland cell clusters and single cells.


Results and conclusion: Bayesian Structural Equation Mod-elling indicated that gene expression in the 3 HPA axis tissues wasoverall influenced by the gut microbiota status (germ-free or con-ventional) and the stress status (acute stress or no stress) of therats. In conclusion, this microgenomics workflow has allowed toaddress the issue of gene expression analysis in a heterogeneousset of tissues involved in a specific neuroendocrine pathway.

“This work was supported by Thermo Fisher Scientific-Arcturuswho kindly provided PicoPure RNA extraction kits, TaqMan

®

PreAmp Master Mix and Cells-to-CTTM kits and TaqMan

®Array

experiments.”


dx.doi.org/10.1016/j.bdq.2017.02.156

BDQ OFC Mockup - Gene-Quantification · 2017-06-26 · qPCR, dPCR & NGS 2017 8th International Gene...

Documents

Transcript of BDQ OFC Mockup - Gene-Quantification · 2017-06-26 · qPCR, dPCR & NGS 2017 8th International Gene...