Targeted Exon Sequencing by In‐Solution Hybrid Selection

Brendan Blumenstiel1, Kristian Cibulskis1, Sheila Fisher1, Matthew DeFelice1, Andrew Barry1, Tim Fennell1, Justin Abreu1, Brian Minie1, Maura Costello1, Geneva Young1, Jared Maquire1, Andrew Kernytsky1, Alexandre Melnikov1, Peter Rogov1, Andreas Gnirke1, Stacey Gabriel1

1 Broad Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 18.4
DOI:  10.1002/0471142905.hg1804s66
Online Posting Date:  July, 2010
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Abstract

This unit describes a protocol for the targeted enrichment of exons from randomly sheared genomic DNA libraries using an in‐solution hybrid selection approach for sequencing on an Illumina Genome Analyzer II. The steps for designing and ordering a hybrid selection oligo pool are reviewed, as are critical steps for performing the preparation and hybrid selection of an Illumina paired‐end library. Critical parameters, performance metrics, and analysis workflow are discussed. Curr. Protoc. Hum. Genet. 66:18.4.1‐18.4.24 © 2010 by John Wiley & Sons, Inc.

Keywords: exon sequencing; hybrid selection; mutation discovery; DNA sequencing; targeting

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: DNA Fragmentation
  • Basic Protocol 2: Paired‐End Library Preparation
  • Basic Protocol 3: Hybrid Selection
  • Basic Protocol 4: Library Quantification by qPCR
  • Support Protocol 1: Read Alignment and Evaluation of Sequence Data
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: DNA Fragmentation

  Materials
  • DNA sample (e.g., see appendix 3B)
  • Nuclease‐free water
  • 70% (v/v) ethanol
  • NanoDrop ND‐1000 spectrophotometer
  • Covaris S‐2 Sample Preparation System
  • VWR circulating chiller
  • Covaris shearing vial (6 × 16−mm AFA fiber vial; cat. no. 520045)
  • 1.5‐ml microcentrifuge tube
  • Agencourt AMPure XP kit (Beckman Coulter, cat. no. A63881)
  • Magnetic separator (DynaMag Spin Magnet, Invitrogen, cat. no. 123‐20D)
  • Additional reagents and equipment for DNA quantitation ( appendix 3D) and agarose gel electrophoresis (unit 2.7)

Basic Protocol 2: Paired‐End Library Preparation

  Materials
  • Illumina Paired End Sample Prep Kit (cat. no. PE‐102‐1001), containing:
    • 10× T4 DNA ligase buffer w/10 mM ATP
    • T4 polynucleotide kinase
    • T4 DNA polymerase
    • Klenow fragment (3′→5′ exo) and Klenow buffer
    • 10 mM dNTP mix
    • 1 mM dATP
    • DNA ligase and 2× buffer
  • Nuclease‐free water
  • Sheared, cleaned DNA sample (see protocol 1)
  • Paired‐end oligo mix (Illumina)
  • 2× Phusion high‐fidelity PCR master mix (Finnzymes, cat. no. F‐531S)
  • PCR primers, 100 µM each:
    • PE1.0: AAT GATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
    • PE2.0: CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
  • 96‐well PCR plate
  • Thermocycler
  • Additional reagents and equipment for cleaning DNA with AMPure beads (see protocol 1), agarose gel electrophoresis (unit 2.7), and DNA quantitation ( appendix 3D)

Basic Protocol 3: Hybrid Selection

  Materials
  • Adapter‐ligated DNA (see protocol 2)
  • 50× Denhardt's solution
  • 20× SSPE
  • Nuclease‐free water
  • 10% SDS
  • 0.5 M EDTA
  • 1.0 mg/ml human Cot‐1 DNA (Invitrogen, cat. no. 15279‐101)
  • 10.0 mg/ml salmon sperm DNA (Invitrogen, cat. no. 15632‐011)
  • Blocking oligos (200 µM each, custom oligos from IDT)
    • Oligo 1.0: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
    • Oligo 2.0: CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
  • 100 ng/µl Biotinylated RNA Oligo Library (Agilent Technologies SureSelect)
  • 20 U/µl Superase‐In RNAse Inhibitor (Applied Biosystems, cat. no. AM2694)
  • Dynabeads M‐280 Streptavidin Beads (Invitrogen, cat. no. 112‐05D)
  • 5 M NaCl
  • 1 M Tris‐Cl
  • 20× SSC
  • 0.1 N NaOH
  • 2× Phusion high‐fidelity PCR master mix (Finnzymes, cat. no. F‐531S)
  • PCR primers, 100 µM each:
    • PE1.0: AAT GATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
    • PE2.0: CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
  • NanoDrop ND‐1000 spectrophotometer
  • Speedvac evaporator
  • 65°C heating block with 1.5‐ml tube holder
  • 96‐well PCR plates
  • 1.5‐ml microcentrifuge tubes
  • Adhesive plate seal
  • 96‐well thermocycler with heated lid
  • 50 ml conical tube
  • Magnetic separator (DynaMag Spin Magnet, Invitrogen, cat. no. 123‐20D)
  • Additional reagents and equipment for DNA quantitation ( appendix 3D) and cleaning DNA with AMPure beads (see protocol 1)

Basic Protocol 4: Library Quantification by qPCR

  Materials
  • 10 nM PhiX Control Library (Illumina, cat. no. 1006471)
  • Nuclease‐free water
  • Target‐selected DNA library (see protocol 3)
  • 2× Brilliant SYBR Green QPCR Master Mix (Stratagene, cat. no. 600548)
  • 1 mM ROX Reference Dye
  • 1.25 µM P5 PCR primer (AATGATACGGCGACCACCGA)
  • 1.25 µM P7 PCR primer (CAAGCAGAAGACGGCATACGA)
  • 384 well MicroAmp Optical Reaction Plate (Applied Biosystems, cat. no. 4326270)
  • MicroAmp Optical Adhesive Film (Applied Biosystems, cat. no. 4311976)
  • ABI 7900HT Real‐Time PCR System with SDS V2.3 software (Applied Biosystems)
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Figures

Videos

Literature Cited

Literature Cited
   Bashiardes, S., Veile, R., Helms, C., Mardis, E.R., Bowcock, A.M., and Lovett, M. 2005. Direct genomic selection. Nat. Methods 2:63‐69.
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   Gnirke, A., Melnikov, A., Maguire, J., Rogov, P., LeProust, E.M., Brockman, W., Fennell, T., Giannoukos, G., Fisher, S., Russ, C., et al. 2009. Solution hybrid selection with ultra‐long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27:182‐189.
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   Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K., Dewar, K., Doyle, M., FitzHugh, W., et al.; International Human Genome Sequencing Consortium. 2001. Initial sequencing and analysis of the human genome. Nature 409:860‐921.
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   Quail, M.A., Kozarewa, I., Smith, F., Scally, A., Stephens, P.J., Durbin, R., Swerdlow, H., and Turner, D.J. 2008. A large genome center's improvements to the Illumina sequencing system. Nat. Methods 5:1005‐1010.
   Raymond, F.L., Whibley, A., Stratton, M.R., and Gecz, J. 2009. Lessons learnt from large‐scale exon re‐sequencing of the X chromosome. Hum. Mol. Genet. 18:60‐64.
   Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain‐terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463‐5467.
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   Sjöblom, T., Jones, S., Wood, L.D., Parsons, D.W., Lin, J., Barber, T.D., Mandelker, D., Leary, R.J., Ptak, J., Silliman, N., et al. 2006. The consensus coding sequences of human breast and colorectal cancers. Science 314:268‐274.
   Thomas, R.K., Nickerson, E., Simons, J.F., Jänne, P.A., Tengs, T., Yuza, Y., Garraway, L.A., LaFramboise, T., Lee, J.C., Shah, K., et al. 2006. Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nat. Methods 12:852‐855.
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