This document discusses advanced next-generation sequencing (NGS) library preparation methods for challenging samples. It introduces NGS applications using various sample types and sizes. Standard NGS library preparation kits have input requirements that cannot accommodate many sample types and quantities. New techniques optimized for sub-nanogram inputs can construct high-quality libraries from as little as 10 picograms of DNA. These methods show consistent conversion across a wide input range and enable applications like ancient DNA analysis and circulating tumor DNA detection from blood. The document focuses on cell-free DNA library preparation challenges and solutions like the QIAGEN cfDNA library kits.
1. Sample to Insight
Advanced NGS Library Prep For Challenging Samples
Nan Fang, Ph.D., MBA
Associate Director Product Development
NGS Universal Solutions, QIAGEN
Advanced NGS library prep for challenging samples
2. Sample to Insight
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
Advanced NGS library prep for challenging samples
3. Sample to Insight
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.
Advanced NGS library prep for challenging samples
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Agenda
NGS library construction for challenging samples
Challenges
Technical solutions
Applications
Focus on cfDNA library construction
1
2
3
4
Advanced NGS library prep for challenging samples
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Lower sequencing cost opens up NGS to more applications
Advanced NGS library prep for challenging samples
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NGS applications: is the only limit our imagination?
Advanced NGS library prep for challenging 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/
More samples……
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NGS applications: is the only limit our imagination?
Advanced NGS library prep for challenging 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
Different samples……
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Demanding sequence information from challenging samples
Limitations in sample quantity and quality
Advanced NGS library prep for challenging samples
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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
Advanced NGS library prep for challenging samples
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The challenge: as input decreases so does the fraction of useful data
Advanced NGS library prep for challenging samples
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
11. Sample to Insight
Chemistry re-optimized for sub-nanogram samples
Advanced NGS library prep for challenging samples
QIAseq Ultralow Input Library Kits
Consistent conversion across a
10,000-fold input range
1 Tube
2 Reactions
70 Minutes
Up to 43 µl input volume
to maximize dilute samples
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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
QIAGEN Webinar 20160927
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QIAseq Ultralow Input Library Kit for as little as 10 –100 pg DNA
Advanced NGS library prep for challenging samples
• 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
Advanced NGS library prep for challenging samples
High mapping rate, high conversion rate, high uniformity
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High SNP detection sensitivity and specificity from 1 ng GIAB
Advanced NGS library prep for challenging samples
1 ng input GIAB DNA , SNP detection with WGS and WES
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Sensitive mutation detection from minimal input amount
Advanced NGS library prep for challenging samples
Comprehensive coverage and high uniformity ensures sensitive mutation detection
cfDNA or FFPE reference
20 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
Advanced NGS library prep for challenging samples
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
Advanced NGS library prep for challenging samples
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Modified QIAseq Ultralow Input Libray Kit: protocol for WGBS library construction
Advanced NGS library prep for challenging samples
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
Advanced NGS library prep for challenging samples
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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
Advanced NGS library prep for challenging samples
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Challenges in cell-free DNA library construction
Advanced NGS library prep for challenging samples
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
Advanced NGS library prep for challenging samples
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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
Advanced NGS library prep for challenging samples
• 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
Advanced NGS library prep for challenging samples
1-step
protocol
35 min
First one-step shot-gun library prep in market—QIAGEN Proprietary Technology
.
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Advanced NGS library prep for challenging samples
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
Advanced NGS library prep for challenging samples
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QIAseq NGS solutions
Advanced NGS library prep for challenging samples
For more information, visit the QIAGEN website:
www.qiagen.com/us/products/ngs/next-gen-sequencing/
30. Sample to Insight
Questions?
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Advanced NGS library prep for challenging samples
Editor's Notes
Drastic decrease in sequencing costs per genome.
To illustrate the nature of the reductions in DNA sequencing costs, each graph also shows hypothetical data reflecting Moore's Law, which describes a long-term trend in the computer hardware industry that involves the doubling of 'compute power' every two years (See: Moore's Law [wikipedia.org]). Technology improvements that 'keep up' with Moore's Law are widely regarded to be doing exceedingly well, making it useful for comparison.
Moore’s law square
~500x
The American College of Medical Genetics and Genomics (ACMG) recently updated their guidelines to support the use of NIPTs as the optimal initial screening test for all pregnant women, regardless of their age or other risk factors. The ACMG recommends informing pregnant women that these tests are "the most sensitive screening option" for some of the most common genetic conditions, including Down syndrome (trisomy 21), Patau syndrome (trisomy 13), and Edwards syndrome (trisomy 18).