Generating Exome Enriched Sequencing Libraries from Formalin‐Fixed, Paraffin‐Embedded Tissue DNA for Next‐Generation Sequencing

Beth A. Marosy1, Brian D. Craig1, Kurt N. Hetrick1, P. Dane Witmer1, Hua Ling1, Sean M. Griffith1, Benjamin Myers1, Elaine A. Ostrander2, Janet L. Stanford3, Lawrence C. Brody4, Kimberly F. Doheny1

1 Center for Inherited Disease Research, McKusick‐Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, Maryland, 2 National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, 3 Program in Prostate Cancer Research, Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, 4 Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 18.10
DOI:  10.1002/cphg.27
Online Posting Date:  January, 2017
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Abstract

This unit describes a technique for generating exome‐enriched sequencing libraries using DNA extracted from formalin‐fixed paraffin‐embedded (FFPE) samples. Utilizing commercially available kits, we present a low‐input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in‐solution‐targeted selection for exons, followed by sequencing using the Illumina next‐generation short‐read sequencing platform. © 2017 by John Wiley & Sons, Inc.

Keywords: FFPE; DNA repair; DNA library preparation; whole exome sequencing; next‐generation sequencing

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Quantitation and Quality Assessment of FFPE DNA
  • Basic Protocol 2: DNA Repair
  • Basic Protocol 3: Fragmentation
  • Basic Protocol 4: Library Preparation (End Repair, A‐Tailing, Ligation, and Amplification)
  • Basic Protocol 5: Exome Enrichment and Capture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitation and Quality Assessment of FFPE DNA

  Materials
  • Human Genomic DNA Quantification and QC Kit (KapaBiosystems, cat. no. KK4961) containing:
  • Elution buffer (Qiagen, cat. no. 19086)
  • KAPA SYBR FAST qPCR Master Mix (2×):
  • 41‐bp Primer Premix (10×)
  • 129‐bp Primer Premix (10×)
  • 305‐bp Primer Premix (10×)
  • KAPA DNA Standards (1‐5)
  • Ice
  • FFPE‐derived gDNA
  • Nuclease‐free water (ThermoFisher Scientific, cat. no. AM9930)
  • Ice
  • Vortex mixer
  • Microcentrifuge
  • MicroAmp Optical 96‐well Reaction Plate (ThermoFisher Scientific, cat. no. N8010560)
  • MicroAmp Optical Adhesive Film (ThermoFisher Scientific, cat. no. 4311971)
  • Centrifuge
  • qPCR instrument (ABI 7900 or ABI Quantflex)

Basic Protocol 2: DNA Repair

  Materials
  • Agencourt Ampure XP beads (Beckman Coulter, cat. no. A63881)
  • PreCR repair kit (New England Biolabs, cat. no. M0309L) containing:
  • ThermoPol Reaction buffer
  • β‐Nicotinamide adenine dinucleotide (NAD+)
  • PreCR repair mix enzyme
  • Ice
  • FFPE‐derived gDNA sample
  • Nuclease‐free water (ThermoFisher Scientific, cat. no AM9930)
  • dNTP Solution Mix (Enzymatics, cat. no. N2050L)
  • 100% molecular biology grade ethanol
  • Elution buffer (Qiagen, cat. no. 19086)
  • Vortex mixer
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • MicroAmp Optical 96‐well Reaction Plate (ThermoFisher Scientific, cat. no. N8010560)
  • MicroAmp Clear Adhesive Film (ThermoFisher Scientific, cat. no. 4306311)
  • Thermal cycler
  • Pipets
  • DynaMag‐96 Side Skirted Plate Magnet (Invitrogen, cat. no. 12027 or 12331D)

Basic Protocol 3: Fragmentation

  Materials
  • Agencourt Ampure XP (Beckman Coulter, cat. no. A63881)
  • 100% molecular biology grade ethanol
  • Nuclease‐free water (ThermoFisher Scientific, cat. no. AM9930)
  • High Sensitivity DNA kit (Agilent, cat. no. 5067‐4626)
  • 96 MicroTube plate or MicroTube AFA Fiber Snap‐Cap (Covaris, cat. no. 520069 or 520045)
  • Focused‐ultrasonicator (Covaris, model E220)
  • MicroAmp Optical 96‐well Reaction Plate (ThermoFisher Scientific, cat. no. N8010560)
  • Pipets
  • DynaMag‐96 Side Skirted Plate Magnet (Invitrogen, cat. no. 12027 or 12331D)
  • 2100 BioAnalyzer (Agilent)
  • MicroAmp Clear Adhesive Film (ThermoFisher Scientific, cat. no. 4306311)

Basic Protocol 4: Library Preparation (End Repair, A‐Tailing, Ligation, and Amplification)

  Materials
  • Hyper Prep Kit (KapaBiosystems, cat. no. KK8504) containing:
  • End repair/A tailing buffer
  • End repair/A tailing enzyme mix
  • Ligation buffer
  • KAPA T4 DNA ligase
  • KAPA HiFi master mix
  • KAPA primer premix
  • Ice
  • 20% PEG/2.5 M NaCl solution (see recipe)
  • Nuclease‐free water (ThermoFisher Scientific, cat. no. AM9930)
  • Indexed Adapter oligonucleotide (Integrated DNA Technology; see recipe)
  • 100% molecular biology grade ethanol
  • Agencourt Ampure XP beads (Beckman Coulter, cat. no. A63881)
  • High Sensitivity DNA Kit (Agilent, cat. no. 5067‐4626)
  • Elution buffer (Qiagen, cat. no. 19086)
  • Vortex mixer
  • Microcentrifuge
  • MicroAmp Optical 96‐well Reaction Plate (ThermoFisher Scientific, cat. no. N8010560)
  • MicroAmp Clear Adhesive Film (ThermoFisher Scientific, cat. no. 4306311)
  • Thermal cycler
  • DynaMag‐96 Side Skirted Plate Magnet (Invitrogen, cat. no.12027 or 12331D)
  • 2100 BioAnalyzer (Agilent)

Basic Protocol 5: Exome Enrichment and Capture

  Materials
  • DNA libraries
  • Universal Blocker Oligonucleotide (Integrated DNA Technology; see recipe)
  • Indexed Blocker Oligonucleotide (Integrated DNA Technology; see recipe)
  • Nuclease‐free water (ThermoFisher Scientific, cat. no. AM9930)
  • SureSelect XT Human All Exon Kit (Agilent, cat. no. 5190‐8863) containing:
  • SureSelect Hyb #1, 2, 3, 4
  • SureSelect Block # 1, 2
  • RNase Block
  • SureSelect Human All Exon Capture
  • Sure Select Binding Buffer
  • Sure Select Wash Buffer I
  • Sure Select Wash Buffer II
  • Ice
  • Dynabeads My One Streptavidin T1 beads (ThermoFisher Scientific, cat. no. 65602)
  • Agencourt Ampure XP (Beckman Coulter, cat. no. A63881)
  • HiFi HotStart ReadyMix (2×) (KapaBiosystems, cat. no. KK2612)
  • PCR Primer 1 (Integrated DNA Technology; see recipe)
  • PCR Primer 2 (Integrated DNA Technology; see recipe) 100% molecular biology grade ethanol
  • High Sensitivity DNA Kit (Agilent, cat. no. 5067‐4626)
  • MicroAmp Optical 96 well Reaction Plate (ThermoFisher Scientific, cat. no. N8010560)
  • Vacuum concentrator
  • MicroAmp Clear Adhesive Film (ThermoFisher Scientific, cat. no. 4306311)
  • Vortex mixer
  • Microcentrifuge
  • Echotherm heat block (Torrey Pines)
  • Thermal cycler
  • MicroAmp Optical 8‐Cap Strips (ThermoFisher Scientific, cat. no. 4323032)
  • DNA LoBind Tubes (Eppendorf, cat. no. 22431021)
  • DynaMag‐2 Magnet (Invitrogen, cat. no. 12321D)
  • Pipets
  • Tube Rocker BD Clay Adams Nutator Mixer (BD Diagnostics, cat. no. 421105)
  • DynaMag‐96 Side Skirted Plate Magnet (Invitrogen, cat. no. 12027 or 12331D)
  • 2100 BioAnalyzer (Agilent)
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Figures

Videos

Literature Cited

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