Targeted Enrichment of Specific Regions in the Human Genome by Array Hybridization

Catherine Igartua1, Emily H. Turner1, Sarah B. Ng1, Emily Hodges2, Gregory J. Hannon2, Arindam Bhattacharjee3, Mark J. Rieder1, Deborah A. Nickerson1, Jay Shendure1

1 University of Washington, Seattle, Washington, 2 Howard Hughes Medical Institute, Chevy Chase, Maryland, 3 Agilent Technologies, Santa Clara, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 18.3
DOI:  10.1002/0471142905.hg1803s66
Online Posting Date:  July, 2010
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Abstract

While whole‐genome resequencing remains expensive, genomic partitioning provides an affordable means of targeting sequence efforts towards regions of high interest. There are several competitive methods for targeted capture; these include molecular inversion probes, microdroplet‐segregated multiplex PCR, and on‐array or in‐solution capture‐by‐hybridization. Enrichment of the human exome by array hybridization has been successfully applied to pinpoint the causative allele of Mendelian disorders. This protocol focuses on the application of Agilent 1 M arrays for capture‐by‐hybridization and sequencing on the Illumina platform, although the library preparation method may be adaptable to other vendors' array platforms and sequencing technologies. Curr. Protoc. Hum. Genet. 66:18.3.1‐18.3.14 © 2010 by John Wiley & Sons, Inc.

Keywords: resequencing; exome; hybridization; targeted enrichment

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Genomic Enrichment by Array Hybridization
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Genomic Enrichment by Array Hybridization

  Materials
  • Oligos (Table 18.3.1)
  • 1× TE buffer, pH 8.0 (e.g., Fisher BioReagents, cat. no. BP2473‐1; or see appendix 2D)
  • 5 µg of genomic DNA (e.g., see appendix 3C)
  • 10× Next End‐Repair Buffer (New England Biolabs)
  • Next End‐Repair Enzyme Mix (New England Biolabs)
  • Agencourt AMPure XP beads (Beckman Coulter Genomics)
  • 70% (v/v) ethanol
  • 10× PCR buffer with 15 mM MgCl 2 (Applied Biosystems, cat. no. N8080160)
  • 100 mM dATP
  • 5 U/µl Taq DNA polymerase
  • 400 U/µl T4 DNA ligase and 10× T4 ligase buffer (e.g., Enzymatics, cat. no. L603‐HC‐L; http://www.enzymatics.com/)
  • Certified low‐range ultra agarose (e.g., BioRad, cat. no. 161‐3107)
  • 1× TAE buffer: 0.04 M Tris‐acetate, pH 8.0/1 mM EDTA
  • 5× gel loading buffer (e.g., QIAgen, cat. no. 239901)
  • 100‐bp DNA ladder (e.g., New England Biolabs N3231)
  • QIAquick Gel Extraction kit (Qiagen) including:
    • Buffer QG
    • Buffer EB
    • Buffer PE
    • QIAquick spin column
  • Isopropanol
  • 5× Phusion buffer (Finnzymes)
  • 25 mM dNTP mix ( appendix 2D)
  • 2 U/µl Phusion Hot Start polymerase (Finnzymes)
  • 1 mg/ml Cot1 human DNA
  • Oligo aCGH/ChIP‐on‐chip Hybridization Kit (Agilent, cat. no. 5188‐5220) including:
    • 10× Oligo aCGH/ChIP‐on‐Chip Blocking Agent
    • 2× Hi‐RPM Hybridization Buffer
  • Elution buffer (e.g., Qiagen, cat. no. 19086)
  • Oligo aCGH/ChIP‐on‐Chip Wash Buffer 1 (Agilent, cat. no. 5188‐5221)
  • Oligo aCGH/ChIP‐on‐Chip Wash Buffer 2 (Agilent, cat. no. 5188‐5222)
  • 7.5 M ammonium acetate
  • 5 mg/ml glycogen
  • 100% ethanol, cold
  • 75% ethanol, cold
  • 2× iTaq Supermix (BioRad)
  • 1% (v/v) Tween 20 in H 2O
  • Strip tubes (BioRad, cat. no. TBS‐0201)
  • Thermal cycler (for standard PCR)
  • Covaris microtubes and tube plate (http://www.covarisinc.com/)
  • Covaris (E‐series or S‐series) ultrasonicator
  • Half‐skirted 300‐µl 96‐well plates
  • Standard 96‐well plates
  • Centrifuge with rotor adapted for 96‐well plates
  • Magnetic plate (Dynal MPC‐96 S; Invitrogen, cat. no. 120.27)
  • Comb (to accommodate 40 µl sample/well): 1 mm (d) × 15 mm (w) × 10 mm (h)
  • UV light box
  • Razor blades
  • 15‐ml conical centrifuge tubes
  • 96‐well PCR plate
  • Agilent DNA 1000 Bioanalyzer
  • 1 M DNA Capture Microarray (Agilent, PN: G3358A, AMADID: 027616)
  • DNA Microarray Hybridization Chamber (Agilent)
  • DNA Microarray Hybridization Oven (Agilent)
  • SecureSeal Hybridization Chambers (Grace Bio Labs, cat. no. 621506) including:
    • SecureSeal Chambers
    • SecureSeal Tabs (also sold separately, cat. no. 629200)
  • Heat block (with thermometer)
  • Thermal cycler (for RT‐PCR; e.g., BioRad)
  • Additional reagents and equipment for quantifying DNA using PicoGreen or NanoDrop assay ( appendix 3D) and agarose gel electrophoresis (unit 2.7)
    Table 8.3.1   MaterialsOligonucleotide Sequences for Genome Enrichment by Array Hybridization

    Name Sequence a
    EXOME_BULK_ FWD ACA CTC TTT CCC TAC ACG ACG* C
    EXOME_BULK_ REV CTC GGC ATT CCT GCT GAA C*C
    EXOME_BLOCK_F CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG ATC* T
    EXOME_BLOCK_R AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC* T
    EXOME_BLOCK_F_rev AGA TCG GAA GAG CGT CGT GTA GGG AAA GAG TGT
    EXOME_BLOCK_R_rev AGA TCG GAA GAG CGG TTC AGC AGG AAT GCC GAG
    EXOME_SEQ_AMP_FWD CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG ATC* T
    EXOME_SEQ_AMP_REV AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC* T
    EXOME_ADAPT_HI ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC*T
    EXOME_ADAPT_LO /5Phos/GAT CGG AAG AGC GGT TCA GCA GGA ATG CCG AG

     aAsterisks represent phosphothioate bonds.
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Figures

Videos

Literature Cited

Literature Cited
   Albert, T.J., Molla, M.N., Muzny, D.M., Nazareth, L., Wheeler, D., Song, X., Richmond, T.A., Middle, C.M., Rodesch, M.J., Packard, C.J., Weinstock, G.M., and Gibbs, R.A. 2007. Direct selection of human genomic loci by microarray hybridization. Nat. Methods 4:903‐905.
   Gnirke, A., Melnikov, A., Maguire, J., Rogov, P., LeProust, E.M., Brockman, W., Fennell, T., Giannoukos, G., Fisher, S., Russ, C., Gabriel, S., Jaffe, D.B., Lander, E.S., and Nusbaum, C. 2009. Solution hybrid selection with ultra‐long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27:182‐189.
   Hodges, E., Xuan, Z., Balija, V., Kramer, M., Molla, M.N., Smith, S.W., Middle, C.M., Rodesch, M.J., Albert, T.J., Hannon, G.J., and McCombie, W.R. 2007. Genome‐wide in situ exon capture for selective resequencing. Nat. Genet. 39:1522‐1527.
   Hodges, E., Rooks, M., Xuan, Z., Bhattacharjee, A., Benjamin Gordon, D., Brizuela, L., Richard McCombie, W., and Hannon, G.J. 2009a. Hybrid selection of discrete genomic intervals on custom‐designed microarrays for massively parallel sequencing. Nat. Protoc. 4:960‐974.
   Hodges, E., Smith, A.D., Kendall, J., Xuan, Z., Ravi, K., Rooks, M., Zhang, M.Q., Ye, K., Bhattacharjee, A., Brizuela, L., McCombie, W.R., Wigler, M., Hannon, G.J., and Hicks, J.B. 2009b. High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing. Genome Res. 19:1593‐1605.
   Okou, D.T., Steinberg, K.M., Middle, C., Cutler, D.J., Albert, T.J., and Zwick, M.E. 2007. Microarray‐based genomic selection for high‐throughput resequencing. Nat. Methods 4:907‐909.
   Porreca, G.J., Zhang, K., Li, J.B., Xie, B., Austin, D., Vassallo, S.L., LeProust, E.M., Peck, B.J., Emig, C.J., Dahl, F., Gao, Y., Church, G.M., and Shendure, J. 2007. Multiplex amplification of large sets of human exons. Nat. Methods 4:931‐936.
   Turner, E.H., Ng, S.B., Nickerson, D.A., and Shendure, J. 2009a. Methods for genomic partitioning. Annu. Rev. Genomics Hum. Genet. 10:263‐284.
   Turner, E.H., Lee, C., Ng, S.B., Nickerson, D.A., and Shendure, J. 2009b. Massively parallel exon capture and library‐free resequencing across 16 genomes. Nat. Methods 6:315‐316.
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