target capture of dna from ffpe samples— recommendations for generating robust sequencing data

Kristina Giorda PhD, Staff Scientist

Target capture of DNA from FFPE samples—recommendations for generating robust sequencing data

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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?

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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

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

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

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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

Outline

• Review – Oncology molecular profiling and FFPE tissue– FFPE extraction and gDNA QC methods– Library preparation and target enrichment


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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 )

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Sample

Removeparaffin

Lyse

Heat

Column-basedDNApurification

Bead-basedDNApurification

FFPE QC methods—TapeStation® Instrument (Agilent)

http://www.agilent.com/cs/library/applications/5991-5258EN.pdf9

DNAIntegrityNumber(DIN)

FFPE QC methods—hgDNA Quantification and QC Kit (KAPA)

https://www.kapabiosystems.com/assets/KAPA_hgDNA_Quantification_and_QC_Kit_TDS.pdf 10

Outline



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Library construction

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Fragmentation

EndrepairandA-tailing

Adapterligation

Beadcleanup

Libraryamplification

Beadcleanup

• IDT xGen® Lockdown® Probes– Individually synthesized– Individual QC for every probe– Individually normalized– Pooled

NGS target capture enrichment

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Target enrichment via hybridization

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xGen® Lockdown® Probes are individually synthesized and QCed

Each xGen® Lockdown® Probe receives an individual ESI-MS analysis15

Failed Remade

Fulllength

Truncated

Fulllength

Outline



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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)

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Extraction and purification detailsQIAGEN 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

18Parenthesesshowrecommendedranges

FFPE fixation impacts DNA quality

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DNAyieldandDINwereconsistentforeachFFPEblock

QC methods are consistent

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SampleDINandqualityscoreswereconsistentforeachblock

Qual

ity s

core

(Q12

9/Q4

1)

• 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

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Library modifications

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FFPEsampleswereshearedwith200bp conditionstoachieve300bp inserts

Post-shearingFragmentation


Adapterligation

Beadcleanup


Beadcleanup


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Input 10 ng of library

gDNA 11 cycles

FFPE 12 cycles

Pre-capturePCRamplification:

Input 10 ng of library

Adapter stock 4 µM

Adapter:insert(300 bp)

400:1

Adapterconcentrationused:Fragmentation


Adapterligation

Beadcleanup


Beadcleanup

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

Maximum mean coverage

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Effect of sample quality on maximum mean coverage

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QCresultspredictmaximummeancoverage

Maxm

eancoverage(reads)

Qualityscore(Q129/Q41) DIN

• 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

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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-capturePCRamplificationcycles:

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

Adapterconcentrationsused:

Achievable coverage depth based on sample quality and quantity

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AdjustinginputmassintolibraryconstructioncancompensateforDNAquality

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

Maxm

eancoverage(reads)

Achievable coverage depth based on sample quality and quantity

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Maxm

eancoveragewith

50

nginpu

t(reads)

Minim

uminpu

tfor

meancoverage=500

X(ng)

AdjustinginputmassintolibraryconstructioncancompensateforDNAquality

• 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

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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

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target capture of dna from ffpe samples— recommendations for generating robust sequencing data

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