NGS, omics and applied bioinformatics at CVI · 2011. 9. 16. · NGS, omics and applied...

NGS, omics and applied bioinformatics at CVI CVI Bioinformatics practise, tips and tricks…

Alex Bossers, Freddy de Bree, and Frank Harders et al.

Department of Infection Biology

PathogenOmics group

Nbic::Bioassist 16 September 2011

www.cvi.wur.nl

CVI - Lelystad

Part of WUR – Animal Sciences Group CVI ~250 employees Co-location ASG-Lifestock Research

www.cvi.wur.nl

CVI - Lelystad

Animal disease control Prevention of transmission to humans

Diagnoses and crisis organization Development of animal models and methods/pathobiology Development of models (epidemiology) Development of diagnostic tests Development of intervention tools (vaccines, therapeutics) Pathobiology, animal models and clinical studies

www.cvi.wur.nl

CVI – Department of Infection Biology

Host-pathogen interactions OMICS:

• Proteome / transcriptome / metabolome Immunology and pathogenesis

Vaccine and diagnostics development

Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011

Bioinformatics at CVI Applied bioinformatics

CVI: Frank, Freddy, Alex Small base so collaborations are crucial

• Life stock Research (ASG) and Plant Research International (PRI) • AMC, Uni Manchester, Uni Maryland/NBACC, Roslin, Sanger, and others…

Current/past focus Reversed vaccinology approaches (sequence/knowledge-based vaccine design) NGS CGH/tiling array approaches (molecular epidemiology) In situ proteome array approaches Pathogen genome sequencing and reconstruction Transcriptomics and analysis HT genotyping and automation

Future focus (2012-)

Virusdiscovery and pathogen identification (NGS and DEFRA biochip :: EpiZone chip) NGS transcriptomics vs arrays Pathogen detection/typing solutions (arrays (solid/solution/tubes)) Metagenomics (NGS)


NGS - CVI

CVI: Sanger capillary sequencing and array platforms

Roche 454

Sequencing unit AMC (Amsterdam)

Onderstepoort Veterinary Institute (South Africa)

Illumina at ServiceXS and BaseClear (Leiden)

November 2011: Illumina MiSeek


Pathogens - focus

Coxiella burnetii (Q fever) Streptococcus suis

ESBLs in several species others...

Blue Tongue (BTV) Avian influenza AngHV1 (eel) Prions/TSEs others…

Knowledge-based vaccine design

Reversed vaccinology approaches Sequence (knowledge) based vaccine design Vaccine-candidate reduction

• In silico

– Localization – Motifs for post-translational modifications and immunomodulation

• In vitro by proteome approaches – 2D, SELDI/mass-spec, Western blots,… – In situ protein/proteome arrays

Knowledge building host::pathogen interactions • Transcriptome of host and pathogen


Reversed vaccinology

Genome 2-4Mb

Proteome 2-4k ORFs

In silico

approaches

2D

SELDI

Localization

Predict exposed antigens

Motifs for modification (lipo)

In situ protein arrays

Candidate reduction for vaccines/diagnostics

Genome variation/plasticity

ReversedReversed

Genome4Mb

Reversed

Genome2-4Mb


NGS analysis flow

Raw NGS data 454 shotgun, PE 3kb

Illumina mp50, PE100

BowtieMapping

Filter data

FastQC


Bowtie mapping to check/filter contaminants


NGS analysis flow



BowtieMapping

Gap closure

Filter data

de novo assembly

Scaffolding contigs

FastQC

Newbler (454)

MIRA (mixed)

ABYSS (mixed)

MUMmer / BLA(S)T (reference)

SSPACE (PE data)

IMAGE (PE data)


NGS analysis flow



BowtieMapping

Gap closure

Filter data

de novo assembly

Scaffolding contigs

FastQC

Newbler (454)

MIRA (mixed)

ABYSS (mixed)

MUMmer / BLA(S)T (reference)

SSPACE (PE data)

IMAGE (PE data) Comparisons (Mumer)


Genome comparisons (MUMmer)


Genome annotation (visualised Artemis)


CVI (galaxy) servers


Tool versioning

Subversion repositories Personal Galaxy

• Development server • Production server • Project specific galaxy modifications

Assembla issue/bug/feature tracking and ticketing system

Wiki for developers

Galaxy toolshed


Tool versioning

Subversion repositories Personal Galaxy

• Development server • Production server • Project specific modified galaxy instance(s)

Assembla.com bug/feature tracking and ticketing system

Wiki for developers

Galaxy toolshed


Software tools Notes

Evernote ~ electronic journal (free win/mac/android/linux) Doit.im for Getting Things Done (free all major platfoms) Freemind mindmapping ManicTime (time tracking)

Version/source control Subversion repositories and ticketing at Assembla Eclipse (IDE for perl, MySQL, R, some php) PyCharm / PHPStorm (python/PHP IDE from jetbrains) dbWrench (DB administration)

Linux command line Bash / sed / grep

Others R Galaxy



Evern

ote

exam

ple

(Lin

ux N

evern

ote

)

Favourite websites

Galaxy Tool syntax http://wiki.g2.bx.psu.edu/Admin/Tools/Tool%20Config%20Syntax

PerlMonks

BioStar.stackoverflow.com (ALchEmiXt)

SeqAnswers.com

CodeIgniter PHP framework (MVC)

Assembla.com

Ubuntu server guide

Geek and Poke (http://geekandpoke.typepad.com)


Challenges and upcoming work

Work Only very limited datasets yet Complete several genomes for publication Improve reversed vaccinology tools

• Annotations/motifs • Easy filtering differences

Link galaxy directly to Artemis and ACT Challenges

Let agent expert do the work Virusdiscovery NGS transcriptomics (RNAseq)

STORAGE!


NGS, omics and applied bioinformatics at CVI · 2011. 9. 16. · NGS, omics and applied...

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