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Target capture of DNA from FFPE samples— recommendations for generating robust sequencing data
The document provides recommendations for optimizing the capture and sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) samples, emphasizing the importance of sample quality evaluation and extraction methods. It outlines an experimental approach with phases that assess the variability of extraction kits, the predictiveness of quality control methods, and the feasibility of constructing high-quality capture libraries from FFPE samples. Additionally, it discusses the impact of sample quality and input amounts on library preparation and sequencing performance.
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Target capture of DNA from FFPE samples— recommendations for generating robust sequencing data
- 1. Kristina Giorda PhD,Staff Scientist Target capture of DNA from FFPE samples—recommendations for generating robust sequencing data 1
- 2. Outline • Review – Oncologymolecular profiling and formalin-fixed, paraffin-embedded (FFPE) tissue – FFPE extraction and gDNA QC methods – Library preparation and target enrichment • Experimental approach – Phase 1—Do FFPE extraction kits or QC methods vary? – Phase 2—Are QC methods predictive of library quality? – Phase 3—Can high quality capture libraries be made with FFPE samples? 2
- 3. Precision health andoncology • White House Precision Health Initiative mission statement – To enable a new era of medicine through research, technology, and policies that empower patients, researchers, and providers to work together toward development of individualized care. • National Cancer Institute defines precision medicine as – Discovering unique therapies that treat an individual’s cancer based on the specific abnormalities of their tumor. From www.cancer.gov
- 4. Mutation profiles mayinform cancer treatment Li T, Kung HJ, et al. (2013) Genotyping and genomic profiling of non-small-cell lung cancer: Implications for current and future therapies. J Clin Oncol, 31(8):1039–1049. 4
- 5. FFPE tumor tissue •Preferred method for tissue preservation in clinical practice • Routinely used for multiple analyses methods, including immunohistochemistry (IHC), in situ hybridization, and next generation sequencing • Notorious for suboptimal DNA quantity and quality • Sample quality evaluation is key to optimizing downstream processing 5
- 6. Variable DNA yieldand quality from FFPE blocks Arreaza G, Qiu P, et al. (2016) Pre-analytical considerations for successful next-generation sequencing (NGS): Challenges and opportunities for formalin-fixed and paraffin-embedded tumor tissue (FFPE) samples. Int J Mol Sci, 17(9):1579. 6
- 7. Outline • Review – Oncologymolecular profiling and FFPE tissue – FFPE extraction and gDNA QC methods – Library preparation and target enrichment • Experimental approach – Phase 1—Do FFPE extraction kits or QC methods vary? – Phase 2—Are QC methods predictive of library quality? – Phase 3—Can high quality capture libraries be made with FFPE samples? 7
- 8. FFPE sample extractionmethods • Remove paraffin with QIAGEN Deparaffinization Solution • FFPE extraction options – QIAamp® DNA FFPE Tissue Kit (column-based; QIAGEN ) – ReliaPrep™ FFPE gDNA Miniprep System (column-based; Promega ) – E.Z.N.A® FFPE DNA Kit (column-based; Omega Bio-tek ) – Mag-Bind® FFPE DNA Kit (bead-based; Omega Bio-tek ) 8 Sample Remove paraffin Lyse Heat Column-based DNA purification Bead-based DNA purification
- 9. FFPE QC methods—TapeStation®Instrument (Agilent) http://www.agilent.com/cs/library/applications/5991-5258EN.pdf 9 DNA Integrity Number (DIN)
- 10. FFPE QC methods—hgDNAQuantification and QC Kit (KAPA) https://www.kapabiosystems.com/assets/KAPA_hgDNA_Quantification_and_QC_Kit_TDS.pdf 10
- 11. Outline • Review – Oncologymolecular profiling and FFPE tissue – FFPE extraction and gDNA QC methods – Library preparation and target enrichment • Experimental approach – Phase 1—Do FFPE extraction kits or QC methods vary? – Phase 2—Are QC methods predictive of library quality? – Phase 3—Can high quality capture libraries be made with FFPE samples? 11
- 12.
- 13. • IDT xGen®Lockdown® Probes – Individually synthesized – Individual QC for every probe – Individually normalized – Pooled NGS target capture enrichment 13
- 14. Target enrichment viahybridization 14
- 15. xGen® Lockdown® Probesare individually synthesized and QCed Each xGen® Lockdown® Probe receives an individual ESI-MS analysis 15 Failed Remade Full length Truncated Full length
- 16. Outline • Review – Oncologymolecular profiling and FFPE tissue – FFPE extraction and gDNA QC methods – Library preparation and target enrichment • Experimental approach – Phase 1—Do FFPE extraction kits or QC methods vary? – Phase 2—Are QC methods predictive of library quality? – Phase 3—Can high quality capture libraries be made with FFPE samples? 16
- 17. Extraction kit comparison •Do FFPE extraction kits or QC methods vary? • DNA was isolated from 5 FFPE blocks – 10 µm scrolls for each extraction – 4 different kits: • QIAamp® DNA FFPE Tissue Kit (column-based; QIAGEN ) • ReliaPrep™ FFPE gDNA Miniprep System (column-based; Promega ) • E.Z.N.A® FFPE DNA Kit (column-based; Omega Bio-tek ) • Mag-Bind® FFPE DNA Kit (bead-based; Omega Bio-tek ) – Samples were assessed with Qubit® dsDNA BR Assay (Thermo Fisher), TapeStation® instrument (Agilent), and the hgDNA Quantification and QC Kit (Kapa Biosystems) 17
- 18. Extraction and purificationdetails QIAGEN Promega Omega-column Omega-beads Deparaffinization 160 µL/56 oC/3 min Lysis, Proteinase K 56oC/1 hr 56oC/1 hr 55oC/3 hr 55oC/3 hr (3–5 hr) Reverse crosslinking 90oC/60 min 80oC/60 min 90oC/30 min (10–30 min) 90oC/45 min (45–60 min) RNase A 200 µg/RT/ 2 min 40 µg/RT/ 5 min 200 µg/RT/ 5 min 100 µg/RT/ 5 min DNA cleanup Column Beads Elution 60 µL 18 Parentheses show recommended ranges
- 19. FFPE fixation impactsDNA quality 19 DNA yield and DIN were consistent for each FFPE block
- 20. QC methods areconsistent 20 Sample DIN and quality scores were consistent for each block Qualityscore(Q129/Q41)
- 21. • Do FFPEextraction kits or QC methods vary? – FFPE fixation impacts DNA quality – DNA yield and QC methods were consistent for each FFPE block • Are QC methods predictive of target capture performance? – Library construction modifications for FFPE samples • Fragmentation optimization • Pre-capture PCR amplification – Effect of sample quality on library quality using fixed 10 ng input into library construction Extraction kit comparison 21
- 22. Library modifications 22 FFPE samples were sheared with 200 bp conditions to achieve 300 bpinserts Post-shearing Fragmentation End repair and A-tailing Adapter ligation Bead cleanup Library amplification Bead cleanup
- 23. Library modifications 23 Input 10ng of library gDNA 11 cycles FFPE 12 cycles Pre-capture PCR amplification: Input 10 ng of library Adapter stock 4 µM Adapter:insert (300 bp) 400:1 Adapter concentration used:Fragmentation End repair and A-tailing Adapter ligation Bead cleanup Library amplification Bead cleanup
- 24. Target capture enrichment •xGen® Acute Myeloid Leukemia Cancer Panel – Target regions within 260 genes (1.2 Mb target area) • 500 ng barcoded library per capture https://www.idtdna.com/pages/products/nextgen/target-capture/xgen-lockdown-panels/xgen-aml- cancer-panel 24
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- 26. Effect of samplequality on maximum mean coverage 26 QC results predict maximum mean coverage Max mean coverage (reads) Quality score (Q129/Q41) DIN
- 27. • Do FFPEextraction kits or QC methods vary? – FFPE fixation impacts DNA quality – DNA yield and QC methods were consistent for each FFPE block • Are QC methods predictive of target capture performance? – Library construction modifications for FFPE samples • Increase shearing • Add pre-capture PCR cycles – QC methods predict target capture performance • Can high quality capture libraries be made with FFPE samples? – A mass titration was done with high quality, low quality, and control DNA to determine the impact of quality on maximum mean coverage Quality comparison 27
- 28. Library modifications 28 Input 1ng 5 ng 10 ng 25 ng 50 ng 100 ng gDNA 14 12 11 8 7 6 FFPE 16 14 12 9 8 7 Pre-capture PCR amplification cycles: Input 1 ng 5 ng 10 ng 25 ng 50 ng 100 ng Adapter stock 400 nM 2 µM 4 µM 10 µM 20 µM 20 µM Adapter:insert (300 bp) 400:1 400:1 400:1 400:1 400:1 200:1 Adapter concentrations used:
- 29. Achievable coverage depthbased on sample quality and quantity 29 Adjusting input mass into library construction can compensate for DNA quality Sample name Q129/41 DIN gDNA (control) 1.4 9.35 High quality FFPE > 0.4 > 3.5 Low quality FFPE < 0.2 < 2.5 Max mean coverage (reads)
- 30. Achievable coverage depthbased on sample quality and quantity 30 Max mean coverage with 50 ng input (reads) Minimum input for mean coverage = 500X (ng) Adjusting input mass into library construction can compensate for DNA quality
- 31. • Quality andyield of DNA extracted from FFPE samples are likely influenced by preservation and fixation • QC methods predict final library complexity • Increased shearing and added pre-capture PCR cycles were required for FFPE samples • It is important to perform QC on FFPE DNA to determine the minimum input required for deep coverage • Sequencing depth is dependent on DNA quality and input amount FFPE recommendations 31
- 32. Conclusions • xGen® Lockdown®Probes are compatible with varying quantity and quality starting material, allowing analysis of clinically relevant samples • Modified library preparation methods enable deep coverage facilitating accurate detection of mutations with 500X median target coverage 32
- 33. “Best tech support ever, @idtdna!” Questions? TALKTO A PERSON. Lauren SakowskiOur experts are available for consultation. “The people at @idtdna are awesome. A+ for customer service.” Nikolai Braun Contact us by web chat, email, or phone. Find local contact details at: www.idtdna.com Or email: applicationsupport@idtdna.com
- 34. THANK YOU! We willemail you the webinar recording and slides next week.