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


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Agenda







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Microbiome and metagenomic terminology


• 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


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


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


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


Sample to Insight

Agenda







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• 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


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


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


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 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)


Sample to Insight

QIAGEN solution for 16S rRNA: dramatically faster library prep


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


Sample to Insight

Agenda







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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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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.


Sample to Insight

Example application: QIAseq FX for urban metagenomics


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


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


Source: Inferring horizontal gene transfer, Wikipedia https://en.wikipedia.org/wiki/Inferring_horizontal_gene_transfer

Sample to Insight


QIAseq

Library Prep

Kit for NGS

SequencingDNA/RNA

extraction



16S rRNA community

profiling



WGS or long-range

PCR/cDNA-seq





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


Sample to Insight

Mechanical-quality fragmentation from an enzymatic workflow

Sample-to-sample fragmentation reproducibility

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200

400

600

5 min 10 min

Ave

rag

e f

rag

me

nt siz

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(bp)

Fragmentation Time

Frag. #1

Frag. #2

Frag. #3

Frag. #4

Customizable fragment size:

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200

400

600

5 min 10 minAve

rag

e f

rag

me

nt siz

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(bp)

Fragmentation time

Input DNA species

Bacterial Mix

Human

250 bp450 bp

1000 bp


Sample to Insight

QIAseq FX exhibits less GC bias than comparable methods

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Norm

aliz

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overa

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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)

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Genome coverage, 1 ng input

QIAGEN QIAseq FX

NEB enzymatic fragmentation

Covaris + standard library prep

Tagmentation


Sample to Insight

Superior genomic coverage from all-enzymatic library prep

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0.02

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0.08

0.1

0.12

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Fra

ctio

n o

f ta

rge

t g

en

om

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Coverage depth (x)

Genomic coverage distribution

QIAseq FX (100 ng)

NEB enzymatic fragmentation (100 ng)

Covaris + library prep (100 ng)

Tagmentation (1 ng)

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No

rma

lize

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ove

rag

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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


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


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


Sample to Insight

Agenda







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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


Sample to Insight


QIAseq

Library Prep

Kit for NGS

SequencingDNA/RNA

extraction



16S rRNA community

profiling



WGS or long-range

PCR/cDNA-seq





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


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.


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


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)


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


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


Sample to Insight

Agenda







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Sample to Insight


QIAseq

Library Prep

Kit for NGS

SequencingDNA/RNA

extraction



16S rRNA community

profiling



WGS or long-range

PCR/cDNA-seq





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


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


Sample to Insight

96-plex adapter barcode design (equivalent to TruSeq HT)


Sample to Insight

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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:

[email protected]

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QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep

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