Advanced NGS Library Prep for Challenging Samples

Sample to Insight

Advanced NGS library prep for challenging samples

Advanced NGS Library Prep For Challenging Samples

Nan Fang, Ph.D., MBAAssociate Director Product DevelopmentNGS Universal Solutions, QIAGEN

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Welcome to the 3-part series - NGS technology overview and applications

“Overview of NGS technologies and innovative NGS library prep methods”

Part 1: Introduction to next-generation sequencing (NGS) technology

Part 2: Innovative NGS library construction technology

Part 3: Advanced NGS library prep for challenging samples


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Legal disclaimer

• 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.


http://www.qiagen.com/

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Agenda

NGS library construction for challenging samples

Challenges

Technical solutions

Applications

Focus on cfDNA library construction

1

2

3

4


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Lower sequencing cost opens up NGS to more applications


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NGS applications: is the only limit our imagination?

http://www.internationalgenome.org/http://www.uk10k.org/https://www.genomicsengland.co.uk/the-100000-genomes-project/https://www.icarbonx.com/en/https://www.technologyreview.com/s/534591/us-to-develop-dna-study-of-one-million-people/

More samples……

http://www.internationalgenome.org/

http://www.uk10k.org/

https://www.genomicsengland.co.uk/the-100000-genomes-project/

https://www.icarbonx.com/en/

https://www.technologyreview.com/s/534591/us-to-develop-dna-study-of-one-million-people/

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NGS applications: is the only limit our imagination?

http://www.nasa.gov/mission_pages/station/research/news/dna_sequencinghttp://spaceflight101.com/iss/kelly-twins-study/http://www.nature.com/nrmicro/journal/v13/n7/full/nrmicro3509.htmlhttp://www.nature.com/nature/journal/vaop/ncurrent/full/nature19792.html

Different samples……

http://www.nasa.gov/mission_pages/station/research/news/dna_sequencing

http://spaceflight101.com/iss/kelly-twins-study/

http://www.nature.com/nrmicro/journal/v13/n7/full/nrmicro3509.html

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature19792.html

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Demanding sequence information from challenging samples

Limitations in sample quantity and quality


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High genomic complexity of sub-nanogram samples

• 1 nanogram of DNA: • 1 ng/6 pg = ~350 human

haploid genomes

• Theoretical max unique sequence depth of ~350x

• 100 picogram of DNA: • >1000 bacterial cells

• ~15 human cells

• ~35 unique copies of the human haploid genome, in theory enough for 30x WGS

Standard low input NGS library prep 10–100 ng

Bacterium Mammalian cell

200 µl Blood

100 ng

10 ng

1 ng

0.1 ng

Typical Sample DNA

Many real-world samples still cannot meet NGS library prep kit requirements despite containing sufficient genomic complexity for many common analyses

ChIP-Seq, Ancient DNA,

LCM

cfDNA from 5 ml plasma

200 µl blood


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The challenge: as input decreases so does the fraction of useful data


Challenging samples:

• Sample quantity

• Sample quality • Degradation • Modification

• Low sample-to-data conversion due to enzymatic inefficiency and loss during lab processing

• Lower than expected sequencing depth across challenging regions

• Low sequence data quality leading to lower % of mapped reads

• High % adapter contamination

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Chemistry re-optimized for sub-nanogram samples


QIAseq Ultralow Input Library Kits

Consistent conversion across a 10,000-fold input range

End polishing 25°C, 30 min65°C, 15 min

Ligation:25°C, 10 min

Cleanup

Minimal amplification

1 Tube

2 Reactions

70 Minutes

Up to 43 µl input volume to maximize dilute samples

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QIAGEN Webinar 20160927

QIAseq Ultralow Input Library Kit: technical principles

End-polishing enzyme mix A unique mixture of 4 different enzymes for efficient end repair + A-addition in one reaction No interference with the ligation step

End-polishing buffer Balanced formulation for maximal activity of all 4 enzymes Additives that increase the effective concentration of the template DNA to ensure high rate of DNA:enzyme interactions even with minimal DNA input

Ligase High-purity DNA ligase, free of nuclease contamination

Ligation Buffer Additives to ensure high ligase activity by maintaining optimal conformation

Adapter Optimized adaptor modification and concentration to minimize adapter-dimer formation

Library amplification Proprietary high-fidelity PCR mix with minimal bias and high efficiency

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QIAseq Ultralow Input Library Kit for as little as 10 –100 pg DNA


• Ultra efficient enzymatic steps

• Minimal % adapter and even G/C coverage

• Ideal for samples limited in quantity and quality

• Flexible: optimized for sub-nanogram, but delivers excellent performance from 10 pg –100 ng +

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High quality library from low-input GIAB sample

High mapping rate, high conversion rate, high uniformity

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High SNP detection sensitivity and specificity from 1 ng GIAB

1 ng input GIAB DNA , SNP detection with WGS and WES

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Sensitive mutation detection from minimal input amount


Comprehensive coverage and high uniformity ensures sensitive mutation detection

cfDNA or FFPE reference20 ng each, Horizon Discovery

Library construction: QIAseq Ultralow Input Library Kit

(8 cycles PCR)

Target enrichment: xGen Pan Cancer Panel (IDT)

Post-capture amplification:

GeneRead Library Amp Kit

Sequencing: MiSeq®

Data analysis: CLC Biomedical Workbench

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Sensitive mutation detection from minimal input amount

Sensitive and accurate mutation detection with moderate sequencing depth

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QIAseq UltraLow Input Library Kit for ChIP-seq

High correlation of normal vs. low input of ChIP DNA

6.9 ng ChIP DNA

0.69 ng ChIP DNA


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Modified QIAseq Ultralow Input Libray Kit: protocol for WGBS library construction

Efficient WGBS library generation from bisulfite-treated DNA

*6 cycles of library PCR amplification

* For details see poster: Fast and Efficient Post-Bisulfite-Seq Library Construction with QIAseq Ultralow Input DNA Library Protocol on QIAGEN website

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Circulating cell-free DNA sequencing for NIPT and cancer detection

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cfDNA sequencing: optimized sample and library preparation

Integrated workflow

cfDNA sample prep from plasma

End polishing

Adaptor ligation

Clean-up/adaptor removal

Library amplification PCR

NGS library

Based on gold standard QIAamp Technology

Based on QIAseq Ultralow Input Library Kit

Market-leading library amplification technology Optional depending on amount of starting material and intended application

PCR-free cfDNA library construction from 10 ng Input


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Challenges in cell-free DNA library construction

Cell-free DNA poses special challenges for library construction

• Low amount of input DNA• 1-100 ng/ml plasma

• cfDNA amounts in plasma are highly variable • from person to person• different also depending on health status in the same person

• Small cfDNA size (majority ~170 bp)

Integrated sample and library prep protocols are required that work both with low-input DNA amount AND also with wide range of DNA input.

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Robust performance over a broad range of input volumes

Evaluation of differernt plasma input volumes / cfDNA input in library prep

9.5 ng cfDNA from 5 ml plasma 5.7 ng cfDNA from 3 ml plasma

3.8ng cfDNA from 2ml plasma 1.9ng cfDNA from 1ml plasma

Library yield 139 nM in 25 µl Library yield 127 nM in 25 µl

Library yield 83 nM in 25 µl Library yield 59 nM in 25 µl


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Comparison of amplified vs. non-amplified cfDNA libraries

Library concentration from 10 ng cfDNA is sufficiently high for sequencing w/o amplification

8 PC

R cy

cles


• 2 nM library concentration sufficient for whole genome sequencing

• For higher amounts of DNA libraries, PCR enrichment with limited number of cycles is required

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Highly specifiic detection of trisomy 21 and 18

25

Highly specific and sensitive detection of trisomy 21 and 18 in NIPT reference samples

• NIPT reference samples (SeraCare SeraSeq™ Trisomy 21 and 18, 12% fetal cfDNA) were processed using the QIAseq cfDNA All-in-One Kit

• Coverage distributions of the two trisomy samples were normalized against each other

• Trisomy 21 and 18 clearly detectable from only 4-6 M reads

Overrepresented chr 18 in sample T18 (12%)

Overrepresented chr 21 in sample T21 (12%)

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QIAseq cfDNA All-in-One Kits for the Ion Torrent™ platform


cfDNA sample prep from plasma

End repair/adapter ligation/ nick repair(up to 56.5 µl cfDNA input)

Cleanup/adapter removal

Library amplification PCR

NGS library

1-step protocol 35 min

First one-step shot-gun library prep in market—QIAGEN Proprietary Technology .

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Donor #1

Donor #3

Donor #2

QIAseq cfDNA All-in-One Kits for the Ion Torrent platform

• Use 50 µl cfDNA sample in library prep

• Double size selection to generate libraries with narrow size distribution (optimized for Ion Torrent clonal amp)

Efficient construction of high quality DNA libraries

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Performance comparison standard vs.1-step library prep protocol

QIAseq cfDNA All-in-One Kit: performance data for the Ion Torrent platform

2-step protocol

All-in-One protocol

Library yield

Libraries prepared from 5ng cfDNA

2-step protocol All-in-One protocol

Sequencing data: quality assessment

2-step protocol All-in-One protocol

No nucleotide bias at the beginning of reads

Comparable percentage of high quality bases


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QIAseq NGS solutions

For more information, visit the QIAGEN website: www.qiagen.com/us/products/ngs/next-gen-sequencing/

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Advanced NGS Library Prep for Challenging Samples

Healthcare

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