Advanced NGS Library Prep for Challenging Samples
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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.
Advanced NGS Library Prep for Challenging Samples
- 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
- 4. Sample to Insight 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
- 5. Sample to Insight Lower sequencing cost opens up NGS to more applications Advanced NGS library prep for challenging samples
- 6. Sample to Insight 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……
- 7. Sample to Insight 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……
- 8. Sample to Insight Demanding sequence information from challenging samples Limitations in sample quantity and quality Advanced NGS library prep for challenging samples
- 9. Sample to Insight 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
- 10. Sample to Insight 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
- 12. Sample to Insight 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
- 13. Sample to Insight 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 +
- 14. Sample to Insight High quality library from low-input GIAB sample Advanced NGS library prep for challenging samples High mapping rate, high conversion rate, high uniformity
- 15. Sample to Insight 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
- 16. Sample to Insight 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
- 17. Sample to Insight Sensitive mutation detection from minimal input amount Advanced NGS library prep for challenging samples Sensitive and accurate mutation detection with moderate sequencing depth
- 18. Sample to Insight 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
- 19. Sample to Insight 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
- 20. Sample to Insight Circulating cell-free DNA sequencing for NIPT and cancer detection Advanced NGS library prep for challenging samples
- 21. Sample to Insight 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
- 22. Sample to Insight 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 .
- 23. Sample to Insight 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
- 24. Sample to Insight 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
- 25. Sample to Insight 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%)
- 26. Sample to Insight 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 .
- 27. Sample to Insight 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
- 28. Sample to Insight 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
- 29. Sample to Insight 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? Thank you for attending! All our solutions from Sample to Insight on: QIAGEN.com Contact QIAGEN Technical Service Call: 1-800-426-8157 for US Call: +49 2103-29-12400 EU Email: techservice-na@QIAGEN.com techservice-eu@QIAGEN.com QIASeq.NGS@QIAGEN.com QIAwebinars@QIAGEN.com Advanced NGS library prep for challenging samples
Editor's Notes
- #6: 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
- #18: ~500x
- #21: 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).