Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.

1. Kristina Giorda PhD, Staff Scientist
Target capture of DNA from FFPE
samples—recommendations for
generating robust sequencing data
1

2. Outline
• Review
– Oncology molecular 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 and oncology
• 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 may inform 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 yield and 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
– Oncology molecular 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 extraction methods
• 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—hgDNA Quantification and QC Kit (KAPA)
https://www.kapabiosystems.com/assets/KAPA_hgDNA_Quantification_and_QC_Kit_TDS.pdf 10

11. Outline
• Review
– Oncology molecular 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. Library construction
12
Fragmentation
End repair and A-tailing
Adapter ligation
Bead cleanup
Library amplification
Bead cleanup

13. • IDT xGen® Lockdown® Probes
– Individually synthesized
– Individual QC for every probe
– Individually normalized
– Pooled
NGS target capture enrichment
13

14. Target enrichment via hybridization
14

15. xGen® Lockdown® Probes are 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
– Oncology molecular 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 purification details
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 impacts DNA quality
19
DNA yield and DIN were consistent for each FFPE block

20. QC methods are consistent
20
Sample DIN and quality scores were consistent for each block
Qualityscore(Q129/Q41)

21. • Do FFPE extraction 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 bp inserts
Post-shearing
Fragmentation
End repair and A-tailing
Adapter ligation
Bead cleanup
Library amplification
Bead cleanup

23. Library modifications
23
Input 10 ng 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

25. Maximum mean coverage
25

26. Effect of sample quality on maximum mean coverage
26
QC results predict maximum mean coverage
Max mean coverage (reads)
Quality score (Q129/Q41) DIN

27. • Do FFPE extraction 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 1 ng 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 depth based 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 depth based 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 and yield 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

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