The Microbiome and Metagenomics Catherine Lozupone CPBS 7711 September 19, 2013.
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep
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Transcript of QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep
Sample to Insight
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep
Jen Fostel, Sr. Global Product Manager
1QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016
Sample to Insight
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• QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
• For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.qiagen.com or can be requested from QIAGEN
Technical Services or your local distributor.
Legal disclaimer
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016
Sample to Insight
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 3
QIAseq Sample to Insight solutions
QIAseq: QIAGEN’s portfolio of Sample to Insight solutions for Illumina and Ion Torrent
sequencers.
From liquid biopsy and oncology biomarker discovery to metagenomics and single cell
sequencing, our application-specific products provide researchers with comprehensive
solutions to deliver the next wave of innovation in the genomics community.
Sample to Insight
Agenda
Introduction to microbial and metagenomics NGS
16S/18S rRNA phylogenomic community profiling
Whole genome and functional metagenomics
NGS from isolated bacterial cells
Wrap up and questions
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 4
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Sample to Insight
Agenda
Introduction to microbial and metagenomics NGS
16S/18S rRNA phylogenomic community profiling
Whole genome and functional metagenomics
NGS from isolated bacterial cells
Wrap up and questions
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 5
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Microbiome and metagenomic terminology
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016
• Human cells are outnumbered by a factor of 10 by microbial cells that live in and on the
human body. These microbial cells comprise the human microbiome.
• Terminology:
o Metagenomics is the study of the collection of genomes derived from a specific sample
or community
o Microbes are microscopic organisms that can be either single or multicellular
o Microbiota are the microbes that live in a specific location (e.g., the human body, the
gut, soil, etc.)
o Microbiome: collective genes of these microscopic co-inhabitants in a system (e.g.,
human host)
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Source: Human mMcrobiome Project, NIH http://commonfund.nih.gov/hmp/index
Sample to Insight
Environmental microbiomes and metagenomics
Multidisciplinary effort to survey the microbial composition of diverse environments across the globe:
• Aims to process 200,000 samples from different biomes and generate a database of
microbes and their gene products
• Estimates of bacterial diversity:
o 160 distinct types of bacteria in 1 ml of ocean water
o 6400–38,000 types of bacteria in 1 gram of soil
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 7
Source: Curtis, T.P., Sloan, W.T. and Scannell, J.W. (2002) Estimating prokaryotic diversity and its limits. Proc Natl Acad Sci USA 99, 10494–9.
These estimates are for bacteria alone; they do not include viruses, archaea or fungi
Sample to Insight
Cataloging microbial communities
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016
Current Methods for Microbial Analysis
• Culture
• Gene cloning (Pan 16S rRNA) and Sanger sequencing
• Microarray
• Next-generation sequencing
o 16S rRNA sequencing
o Whole genome sequencing
• MALDI
• qPCR – Target dependent
o 16S rRNA gene
o Other relevant gene (antibiotic resistance gene, virulence factor gene)
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Sample to Insight
Excellent correlation between qPCR and NGS profiling
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016
Profiles of vaginal flora by qPCR and whole genome sequencing
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Sample to Insight
Choosing technology for microbial analysis and metagenomics
Speed and specificity vs. discovery at scale
QIAseq
NGS
library
prep
QIAGEN
microbial
qPCR
assays
• Highest throughput
• Compatible with both 16S
rRNA-seq and genome-wide
discovery
• Specific, easy-to-set-up, off-
the-shelf assays
• Fastest assay and analysis:
under 3 hours
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 10
Sample to Insight
Agenda
Introduction to microbial and metagenomics NGS
16S/18S rRNA phylogenomic community profiling
Whole genome and functional metagenomics
NGS from isolated bacterial cells
Wrap up and questions
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 11
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Sample to Insight
• 16S rRNA gene as a
phylogenetic marker for
bacterial ID
• Sequence similarity = 95%
genus level, 97% species
level, 99% strain level
• Classification from the variable
sequences
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 12
16S rRNA community profiling
Sample to Insight
Example data: findings from the NIH Human Microbiome Project
Microorganisms cluster by body siteCataloguing efforts by the NIH Human Microbiome Project suggest:
• Different body sites have unique
communities
• Race, age, gender, weight and
ethnicity all have effects on
microbiome populations
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 13
Sources:
Rodrigues Hoffman, A., Proctor, L.M., Surette, M.G., Suchodolski, J.S. (2016) The microbiome: the trillions of microorganisms that maintain health and disease in
humans and companion animals. Vet Pathol 53, 10–21.
Human Microbiome Project, NIH https://commonfund.nih.gov/hmp/index
Human Microbiome Project Consortium. (2012) Structure, function and diversity of the healthy human microbiome. Nature 486: 207–14.
Sample to Insight
Microbial qPCR assays and multiplex PCRs for identification and profiling use probes
and primers against 16S rRNA variable regions.
16S rRNA
PCRLibrary prep Cleanup
Library
amplificationSequencing
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 14
16S rRNA community profiling
Sample to Insight
QIAseq 1-Step Amplicon
QIAseq
NGS library
prep kits
SequencingDNA/RNA
extraction
30-minute, room temperature
library prep for PCR products
16S rRNA community
profiling
QIAseq FX DNA Library KitAll-enzymatic workflow without
Covaris shearing or GC bias
WGS or long-range
PCR/cDNA-seq
QIAseq FX Single Cell DNA
Library Kit From single isolated cells to
libraries in under 4 hoursBacterial single cell WGS
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 15
QIAseq 1-Step Amplicon for the simplest amplicon library prep
Sample to Insight
QIAGEN recomends: QIAseq 1-Step Amplicon Library Kit
30-minute single-tube, room-temperature library prep for amplicon-targeted resequencing
• Proprietary single-tube approach: only one pipetting step
• Fastest possible library prep: 30 minutes, room-temperature incubation
Library amplification (optional)
(45 min)
Single-tube room-temperature library
prep
(30 min)
16S rRNA PCR product
(1–500 ng)
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 16
Sample to Insight
QIAGEN solution for 16S rRNA: dramatically faster library prep
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 17
Sample to Insight
QIAseq 1-Step Amplicon Library Kit: contents and advantages
Kits contain:
• Enzymes and buffers for 1-step
library preparation
• Single-use, 96-plex Illumina
adapters (96-reaction format only)
• HiFi polymerase and buffer for
library amplification (optional)
What’s not included:
• Agencourt AMPure XP beads for reaction
cleanup
• qPCR reagents for library quantification:
recommended for accurate flow cell loading,
especially for PCR-free workflows.
Fast and
simple
library prep
Replace 2–3
hours of
sequential
steps with a
single 30-
minute reaction
Room temperature
library prep now
completely on the
benchtop, perfect
for automation
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 18
Sample to Insight
Agenda
Introduction to microbial and metagenomics NGS
16S/18S rRNA phylogenomic community profiling
Whole genome and functional metagenomics
NGS from isolated bacterial cells
Wrap up and questions
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 19
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Sample to Insight
NGS whole genome and functional metagenomics
Complexity and function of genomic content – function of microbiome enables individual survival
• Genetic content development in each
organism for its own survival in a specific
environment
• Metabolism tuned to local nutrient sources
• Virulence factors for stable colonization
• Antibiotic resistance genes to metabolize
toxins
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QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 20
Image source: Kyoto Encyclopedia of Genes and Genomes (KEGG) http://www.kegg.jp/kegg/
Sample to Insight
Example application: antibiotic resistance detection
Antibiotic resistance genes in our food supply?
• One potential source of acquiring antibiotic
resistance genes is through the food supply.
• Both livestock and feed may acquire
antibiotic resistant bacteria through different
mechanisms.
• Food can be exposed to antibiotic resistant
bacteria through fertilizer originating from
wastewater treatment plants. This exposure,
in addition to increasing administration of
antibiotics to livestock, can lead to food
becoming a potential source of antibiotic-
resistant genes.
• Consequently, a horizontal gene transfer to
pathogenic enteropathogens may result –
leading to drug resistance in humans. This
dynamic highlights the importance of
surveillance and prevention of antibiotic-
resistant genes in food.
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 21
Sample to Insight
Example application: QIAseq FX for urban metagenomics
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 22
Sample to Insight
Challenge in whole genome metagenomics: DNA fragmentation
Mechanical shearing Tagmentation
Fragment
DNA
Add
adapters
Amplify and
QC
library
Typical WGS
library prep
process:
• Higher costs
• Harder to scale up
• Labor-intensive
• Strong GC bias
• Inflexible input requirement
• Inflexible fragment size
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 23
Sample to Insight
Why GC bias makes a difference in metagenomic studies
GC content varies widely within and between microbial genomes
E. coli
B. pertussis
F. nucleatum (low GC)
• Tagmentation cleaves low GC sequences preferentially
• Sequence preference strongly biases species/region detection
Tagmentation fragment size: 1 ng DNA
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 24
Source: Inferring horizontal gene transfer, Wikipedia https://en.wikipedia.org/wiki/Inferring_horizontal_gene_transfer
Sample to Insight
QIAseq 1-Step Amplicon
QIAseq
Library Prep
Kit for NGS
SequencingDNA/RNA
extraction
30-minute, room temperature
library prep for PCR products
16S rRNA community
profiling
QIAseq FX DNA Library KitAll-enzymatic workflow without
Covaris shearing or GC bias
WGS or long-range
PCR/cDNA-seq
QIAseq FX Single Cell DNA
Library Kit From single isolated cells to
libraries in under 4 hoursBacterial single cell WGS
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 25
QIAseq FX for whole genome and functional metagenomics
Sample to Insight
QIAGEN solution: all-enzymatic nuclease-based library prep
FX chemistry for random, enzymatic DNA fragmentation included in QIAseq FX Kits:
adapter ligation
(45 min)
QIAGEN HiFi library amp
(45 min – optional)
Single-tube FX reaction
(50–60 min)
Purified gDNA
1 ng – 1 µg
Ready-to-sequence library
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 26
Sample to Insight
Mechanical-quality fragmentation from an enzymatic workflow
Sample-to-sample fragmentation reproducibility
0
200
400
600
5 min 10 min
Ave
rag
e f
rag
me
nt siz
e
(bp)
Fragmentation Time
Frag. #1
Frag. #2
Frag. #3
Frag. #4
Customizable fragment size:
0
200
400
600
5 min 10 minAve
rag
e f
rag
me
nt siz
e
(bp)
Fragmentation time
Input DNA species
Bacterial Mix
Human
250 bp450 bp
1000 bp
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 27
Sample to Insight
QIAseq FX exhibits less GC bias than comparable methods
0
0.5
1
1.5
2
0 20 40 60 80 100
Norm
aliz
ed c
overa
ge
GC% over 100 bp regions
Genome coverage , 100 ng input
QIAGEN QIAseq FX
NEB enzymatic fragmentation
Covaris + standard library prep
(Tagmentation not possible at
100 ng input)
0
0.5
1
1.5
2
0 20 40 60 80 100
GC% over 100 bp regions
Genome coverage, 1 ng input
QIAGEN QIAseq FX
NEB enzymatic fragmentation
Covaris + standard library prep
Tagmentation
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 28
Sample to Insight
Superior genomic coverage from all-enzymatic library prep
0
0.02
0.04
0.06
0.08
0.1
0.12
0 20 40 60 80 100
Fra
ctio
n o
f ta
rge
t g
en
om
e
Coverage depth (x)
Genomic coverage distribution
QIAseq FX (100 ng)
NEB enzymatic fragmentation (100 ng)
Covaris + library prep (100 ng)
Tagmentation (1 ng)
0
0.5
1
1.5
2
0 20 40 60 80 100
No
rma
lize
d c
ove
rag
e
GC% over 100 bp regions
Coverage by GC%
QIAseq FX (50 ng PCR-free)
QIAseq FX (100 ng PCR-free)
Covaris + library prep (100 ng) with
PCR
QIAseq FX (100 ng) with PCR*Supplier I does not offer a kit for tagmentation from 100 ng DNA
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 29
Sample to Insight
Case study: QIAseq FX DNA Library from GC-rich gDNA
Paired-end mapping read track
Bacteria: Bordetella pertussis, ~67% GC
Input gDNA amount: 1 ng
Library construction: QIAseq FX
Library size (insert + adaptor): 499 bp
Library amplification: 10 cycles
Data analysis: CLC Genomics Workbench
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 30
Sample to Insight
QIAGEN poster: Phillip Widman at European Microbiome Congress
Cattle fecal samples:
5 affected animals
5 unaffected animals
Johne’s Disease, or paratuberculosis:
• Gastrointestinal disease of
ruminants
• Mycobacterium avium ss
paratuberculosis
• Symptoms: rapid weight loss,
diarrhea
• Diagnosis: PCR, culture
AllPrep
PowerFecal
DNA
Isolation Kit
DNA
16S rRNA amplification
QIAGEN QuantiTect Probe PCR Kit
MiSeq; 500 bp paired-end
Analysis:
CLC Microbial
Genomics Module
Whole genome metagenomics
QIAseq FX DNA Library Kit
MiSeq; 500 bp paired-end
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 31
Sample to Insight
Agenda
Introduction to microbial and metagenomics NGS
16S/18S rRNA phylogenomic community profiling
Whole genome and functional metagenomics
NGS from isolated bacterial cells
Wrap up and questions
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 32
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5
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Sample to Insight
Challenge: extremely low DNA content in isolated bacterial cells
Standard
NGS library
prep input:
100–1000 ng
Bacterium Mammalian cell 200 µl Blood
1 µg
1 ng
1 pg
1 fg
Ave
rag
e D
NA
co
nte
nt
Many “standard” NGS library prep methods still require 100 ng or more input DNA
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 33
Sample to Insight
QIAseq 1-Step Amplicon
QIAseq
Library Prep
Kit for NGS
SequencingDNA/RNA
extraction
30-minute, room temperature
library prep for PCR products
16S rRNA community
profiling
QIAseq FX DNA Library KitAll-enzymatic workflow without
Covaris shearing or GC bias
WGS or long-range
PCR/cDNA-seq
QIAseq FX Single Cell DNA
Library Kit From single isolated cells to
libraries in under 4 hoursBacterial single cell WGS
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 34
QIAseq FX Single Cell kits for NGS from isolated microbial cells
Sample to Insight
QIAGEN Solution: use MDA, not PCR, for whole genome amp
Alkaline
denaturation
Hexamer random
primers
Phi29 polymerase
strand displacement
(30°C)
• Modified high-fidelity Phi29 polymerase (SensiPhi)
o High enzyme processivity – molecular weight product (10–100 kb)
o High-fidelity proofreading activity – 1000-fold higher accuracy than that of Taq
• Extremely high yields (micrograms) from a single cell without PCR
REPLI-g: multiple displacement amplification in QIAseq Single Cell library prep kits
Displaced strand
becomes a template for
replication
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 35
Sample to Insight
Multiple displacement amplification (MDA) by QIAGEN
QIAGEN’s REPLI-g MDA method
• Random primers are extended at 30°C as the
polymerase moves along the gDNA or cDNA
strand – displacing the complementary strand
while becoming a template, itself, for replication.
In contrast to PCR amplification, MDA:
• Does not require thermal cycling, which can
bias products against high GC sequences.
• Has fewer template-binding events but has very
long fragments that effectively replicate the
genome.
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 36
Sample to Insight
MDA amplifies through gDNA (or cDNA) secondary structures
• Denatured gDNA has a complex
secondary structure
• Consists of regions of ssDNA and
dsDNA that can form complicated
hairpins and loops
QIAGEN’s Phi29-based SensiPhi enzyme handles complex DNA structures
to generate extremely long amplicons
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 37
Sample to Insight
Excellent amplification uniformity across wide GC content range
Advantages of coverage
uniformity:
• Better de novo genome assembly
• Wide species applicability
• Lower total read number required;
higher multiplexing
• Advantageous for low-pass
sequencing strategy
1 pg DH10B DNA, amplified with either REPLI-g Single Cell Kit or by
MALBAC; sequenced on MiSeq Illumina (V2, 2 x 150 nt)
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 38
Sample to Insight
QIAseq FX Single Cell DNA Library Kit: Contents
Kits contain:
• Cell lysis reagents
• Enzymes and buffers for whole genome
amplification
• Enzymatic DNA fragmentation
• Single-step NGS library preparation
• Single-use, disposable Illumina adapters in
96-well format
• Multiple reagent aliquots to reduce
contamination risk and freeze-thaw cycles
What’s not included:
• Agencourt AMPure XP beads for library purification
• PCR reagents for library amplification: not needed
as the entire workflow is PCR-free
• qPCR reagents for library quantification:
recommended for accurate flow cell loading
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 39
Sample to Insight
QIAseq FX Single Cell DNA Library Kit: Workflow
Cell lysis
15 min
WGA
2 h
FX library
preparation
70 min
Purification
20 min
Illumina
sequencing
3 h 45 min with ~40 min hands-on time
Cell lysis
• From single eukaryotic or bacterial cells, or from
small amounts (pg – ng) of intact gDNA
• Starting with 4 µl cell material in PBS (included)
Whole genome amplification
• Amplified gDNA can be used directly or frozen until needed
• Excess amplified gDNA can used for PCR followup studies
• Precise quantification of amplified DNA not needed before library prep
NGS library preparation
• All-enzymatic workflow requires only a standard
thermocycler
• Convenient 96-plex plate-format adapters
• Completely PCR-free workflow
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 40
Sample to Insight
Agenda
Introduction to microbial and metagenomics NGS
16S/18S rRNA phylogenomic community profiling
Whole genome and functional metagenomics
NGS from isolated bacterial cells
Wrap up and questions
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 41
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1
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4
Sample to Insight
QIAseq 1-Step Amplicon
QIAseq
Library Prep
Kit for NGS
SequencingDNA/RNA
extraction
30-minute, room temperature
library prep for PCR products
16S rRNA community
profiling
QIAseq FX DNA Library KitAll-enzymatic workflow without
Covaris shearing or GC bias
WGS or long-range
PCR/cDNA-seq
QIAseq FX Single Cell DNA
Library Kit From single isolated cells to
libraries in under 4 hoursBacterial single cell WGS
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 42
The QIAseq portfolio for Metagenomics and Microbiome (Illumina)
Sample to Insight
QIAGEN’s microbial analysis DNA isolation and qPCR products
Sample to Insight: Microbial DNA qPCR Assays and Arrays
QIAsymphony/QIAcube/QIAcube HT QIAgility Rotor-Gene Q
DNA
isolation
Assays
and arrays
Data
analysis
• AllPrep PowerFecal DNA Isolation Kit
• QIAamp DNA Microbiome Kit
• mericon Bacteria Kit
• QIAamp UCP Pathogen Mini Kit
• QIAamp DNA Stool Mini Kit
• QIAamp UCP Pathogen Blood Kit
• QIAamp DNA Mini Kit
• MagAttract HMW DNA Kit
• QIAseq portfolio of library prep kits
• Microbial DNA qPCR Arrays
• Microbial DNA qPCR Assay Kits
• Microbial DNA qPCR Assays
• MicrobialDNA qPCR Multi-Assay Kits
• Custom Microbial DNA qPCR Arrays
• GeneGlobe Data
Analysis Center
• QIAGEN Microbial
Genomics Pro
Suite (NGS)
Sample Insight
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 43
Sample to Insight
QIAseq ligation-based library prep kit contents
End repair and a-addition
• End-polishing enzyme
• End-polishing buffer 10x
Adapter ligation
• Ultralow Input ligase
• Ultralow Input ligase buffer 4x
HiFi PCR Master
Mix
96-plex
adapter
oligos
(96 reaction
size)
Nuclease-free water
Illumina
library amp
PCR primer
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 44
Sample to Insight
96-plex adapter barcode design (equivalent to TruSeq HT)
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 45
Sample to Insight
46
Questions?
Thank you for attending!
All our solutions from Sample to Insight on:
QIAGEN.com
Contact QIAGEN Technical Service
Call: 1-800-362-7737 or 1-800-426-8157 for US
Call: +49 2103-29-12400 EU
Email:
QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016
www.qiagen.com/us/products/ngs/ngs-life-sciences/metagenomics/