Exome Sequencing by Targeted Enrichment

Michael James Clark1, Rui Chen1, Michael Snyder2

1 Department of Genetics, Stanford University School of Medicine, Palo Alto, California, 2 Center for Genomics and Personalized Medicine, Stanford University, Palo Alto, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.12
DOI:  10.1002/0471142727.mb0712s102
Online Posting Date:  April, 2013
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Abstract

This unit describes methods for targeted enrichment of the exon‐coding portions of the genome using Agilent SureSelect Human All Exon 50 Mb and Roche Nimblegen SeqCap EZ Exome platforms. Each platform targets and enriches a large overlapping portion of the greater human exome. The protocols here describe the biochemical procedures used to enrich exomic DNA with each platform, including recommended modifications to the manufacturers' protocols. In addition, a brief description of the sequencing protocol and estimation of the needed amount of sequencing for each platform is included. Finally, a detailed analytical pipeline for processing the subsequent data is described. These protocols focus specifically on human exome sequencing platforms, but can be applied with some modification to other organisms and targeted enrichment approaches. Curr. Protoc. Mol. Biol. 102:7.12.1–7.12.21. © 2013 by John Wiley & Sons, Inc.

Keywords: exome sequencing; high‐throughput sequencing; next‐generation sequencing; target enrichment; bioinformatics

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Exome Enrichment Using the Agilent SureSelect Human All Exon Platform
  • Basic Protocol 2: Exome Enrichment Using the Nimblegen SeqCap EZ Exome Library SR Platform
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Exome Enrichment Using the Agilent SureSelect Human All Exon Platform

  Materials
  • High‐quality human genomic DNA (gDNA, non‐degraded, A 260/A 280 1.8 to 2.0)
  • Quant‐iT dsDNA BR Assay Kit for Qubit fluorometer (Invitrogen, Q32850)
  • Low TE buffer: 10 mM Tris‐Cl, pH 8.0, 0.1 mM EDTA (see appendix 22 for stock solutions)
  • QIAquick PCR Purification Kit (Qiagen, 28104), including 2‐ml spin columns, 2‐ml collection tubes, Buffers PB, PE, and EB, and 3 M sodium acetate
  • pH indicator (for Buffer PB)
  • Isopropanol (required for Qiagen kits)
  • Agilent DNA 1000 Kit (Agilent, 5067‐1504)
  • Nuclease‐free water (not DEPC‐treated, Ambion, AM9930)
  • MinElute PCR Purification Kit (Qiagen, 28004), including spin columns, 2‐ml collection tubes, Buffers PB, PE, and EB, and 3 M sodium acetate
  • Paired‐End Genomic DNA Sample Prep Kit (Illumina, PE‐102‐1001), including DNA ligase, T4 DNA polymerase, Klenow polymerase and exo fragment, T4 PNK, dNTP mix, dATP, buffers, and paired‐end adapter oligo mix
  • AMPure DNA Purification Kit (SPRI beads, Beckman Coulter UK, A29152)
  • 70% (v/v) ethanol, molecular biology grade
  • Agilent High‐Sensitivity DNA Kit (Agilent, 5067‐4626)
  • 100 RXN Genomic DNA Sample Prep Oligo Only Kit (Illumina, PE‐102‐1003), including PCR primers PE 1.0 and 2.0
  • Phusion High‐Fidelity (HF) PCR Master Mix with HF buffer (Finnzymes, F‐531S; NEB, M0531S)
  • SureSelect Human All Exon Kit (Agilent, G3362A‐L), including oligo capture library, RNase Block, Block #1‐3, Hybridization Buffers (Hyb) #1‐4, Binding Buffer, Wash Buffers #1‐2, Elution Buffer, and Neutralization Buffer
  • Dynabeads M‐280 Streptavidin (Invitrogen, 112‐05D)
  • Herculase II Fusion DNA Polymerase (Stratagene, 600677)
  • Paired‐end cluster generation kit: TruSeq PE Cluster Kit v3‐cBot‐HS Kit (Illumina, PE‐401‐3001), including HP3 and HT1 buffers
  • Qubit fluorometer (Invitrogen, Q32857)
  • 1.5‐ml DNA LoBind tubes, PCR clean (Eppendorf, 022431021)
  • Covaris microTUBEs with AFA fibers and snap caps (Covaris, 520045)
  • Covaris S‐series Single‐Tube Sample Preparation System, Model S2 (Covaris)
  • Agilent 2100 Bioanalyzer (Agilent, G2938C) with Expert software (version B.02.02 and B.02.07 or higher)
  • PCR tubes, strip tubes, or plates
  • Thermal cycler (e.g., BioRad DNA Engine PTC‐200 or ABI Veriti Thermal Cycler)
  • Magnetic bead separator (Dynal DynaMag‐2, Invitrogen, 123‐21D)
  • 37°C heat block
  • Vacuum concentrator (Savant SpeedVac)
  • 65°C circulating water bath
  • 96‐well tube plates with optical strip caps (Agilent, 410088 and 401425)
  • Tube‐strip capping tool (Agilent, 410099)
  • 12‐channel multichannel pipetter (e.g., Rainin Pipet‐Light, L12‐20)
  • Double‐adhesive film (e.g., MicroAmp Clear Adhesive Film, Life Technologies, #4306311)
  • Adams Nutator Mixer (BD Diagnostics, 421105)
  • Additional reagents and equipment for determining DNA concentration ( appendix 3D)
NOTE: Wear powder‐free gloves and use sterile, nuclease‐free, aerosol‐barrier pipet tips throughout the procedure.

Basic Protocol 2: Exome Enrichment Using the Nimblegen SeqCap EZ Exome Library SR Platform

  Materials
  • SeqCap EZ Human Exome Library v2.0 (Roche, 4 or 48 reactions)
  • Paired‐End Genomic DNA Sample Preparation Kit (Illumina, PE‐102‐1001)
  • Agencourt AMPure XP Kit (Agencourt, A63880/A63881/A63882)
  • 70% (v/v) ethanol, freshly prepared
  • Buffers EB, PBI, and PE (Qiagen)
  • MinElute PCR Purification Kit (Qiagen, 28004), including spin columns, 2‐ml collection tubes, and buffers
  • Agilent DNA 1000 Kit (Agilent, 5067‐1504)
  • Phusion High‐Fidelity PCR Master Mix with HF Buffer (NEB, Finnzymes, F‐531S/L)
  • PCR‐grade water (Roche, 03 315 843 001)
  • Custom oligonucleotide primers for LM‐PCR and qPCR (Table 7.12.1)
  • QIAquick PCR Purification Kit (Qiagen, 28106)
  • COT Human DNA, fluorometric grade (Roche, 05 480 647 001)
  • Nimblegen SeqCap EZ Hybridization and Wash Kit (Roche, 05634261001 for 24× or 05634253001 for 96×), including 2× Hybridization Buffer, Hybridization Component A, 10× Wash Buffers (I, II, III, and Stringent), and 2.5× Bead Wash Buffer
  • Dynabeads M‐270 Streptavidin (Invitrogen, 653‐05/06)
  • LightCycler 480 2× SYBR Green I Master Mix (Roche, 04 707 516 001)
  • 0.2‐ml PCR tubes or 96‐well PCR plates
  • 1.5‐ml microcentrofuge tubes
  • DynaMag‐2 Magnet with 16 × 1.5‐ml tube holder (Invitrogen, 123‐21D)
  • Vacuum concentrator (SpeedVac, Savant)
  • Agilent 2100 Bioanalyzer (Agilent, G2938C) with Expert software (version B.02.02 or higher)
  • Thermal cycler capable of maintaining 47°C for 64‐72 hr
  • 18‐G needle or smaller
  • Microcentrifuge with multiplate adapters (16,000 × g capable)
  • 95°C heat block
  • 47°C water bath
  • 384‐well qPCR plates with sealing foils (LightCycler 480 Multiwell Plate 384, Roche, 04 729 749 001)
  • LightCycler 480 Instrument II (Roche Applied Science, 05 015 243 001/05 015 278 001) with Absolute Quantification Analysis Module, version 1.5
  • Additional reagents and equipment for DNA purification with MinElute columns and bioanalysis on an Agilent DNA 1000 chip (see protocol 1), and for DNA quantification ( appendix 3D)
    Table 7.2.1   Materials   Custom Oligonucleotide Primers aa,b,c   Custom Oligonucleotide Primers

    Description Concentration (µM) Sequence
    PE‐PRE1 Oligo 100 5′‐AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC* T‐3′
    PE‐PRE2 Oligo 100 5′‐CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG ATC* T‐3′
    PE‐HE1 Oligo 1000 5′‐AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC* T‐3′
    PE‐HE2 Oligo 1000 5′‐CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG ATC* T‐3′
    PE‐POST1 Oligo 100 5′‐AAT GAT ACG GCG ACC ACC GAG A‐3′
    PE‐POST2 Oligo 100 5′‐CAA GCA GAA GAC GGC ATACGAG‐3′
    qPCR NSC‐0237 F 2 5′‐CGC ATT CCT CAT CCC AGT ATG‐3′
    qPCR NSC‐0237 R 2 5′‐AAA GGA CTT GGT GCA GAG TTC AG‐3′
    qPCR NSC‐0247 F 2 5′‐CCC ACC GCC TTC GAC AT‐3′
    qPCR NSC‐0247 R 2 5′‐CCT GCT TAC TGT GGG CTC TTG‐3′
    qPCR NSC‐0268 F 2 5′‐CTC GCT TAA CCA GAC TCA TCT ACT GT‐3′
    qPCR NSC‐0268 R 2 5′‐ACT TGG CTC AGC TGT ATG AAG GT‐3′
    qPCR NSC‐0272 F 2 5′‐CAG CCC CAG CTC AGG TAC AG‐3′
    qPCR NSC‐0272 R 2 5′‐ATG ATG CGA GTG CTG ATG ATG‐3′

     aPrimers are ordered from a custom oligonucleotide vendor (e.g., IDT, http://www.idtdna.com). PE, paired end; HE, hybridization enhancing.
     bAsterisks represent a phosphorothioate bond before the last T, which prevents degradation during storage.
     cImportant: PE‐PRE1, PE‐PRE2, PE‐HE1, and PE‐HE2 must be HPLC‐purified following synthesis.
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Figures

Videos

Literature Cited

   1000 Genomes Project Consortium. 2010. A map of human genome variation from population‐scale sequencing. Nature 467:1061‐1073.
   Adey, A., Morrison, H.G., Xun, X., Kitzman, J.O., Turner, E.H., Stackhouse, B., MacKenzie, A.P., Caruccio, N.C., Zhang, X., and Shendure, J. 2010. Rapid, low‐input, low‐bias construction of shotgun fragment libraries by high‐density in vitro transposition. Genome Biol. 11:R119.
   Bainbridge, M.N., Wang, M., Burgess, D.L., Kovar, C., Rodesch, M.J., D'Ascenzo, M., Kitzman, J., Wu, Y.Q., Newsham, I., Richmond, T.A., Jeddeloh, J.A., Muzny, D., Albert, T.J., and Gibbs, R.A. 2010. Whole exome capture in solution with 3 Gbp of data. Genome Biol. 11:R62.
   Clark, M.J., Chen, R., Lam, H.Y., Karczewski, K.J., Chen, R., Euskirchen, G., Butte, A.J., and Snyder, M. 2011. Performance comparison of exome DNA sequencing technologies. Nat. Biotechnol. 29:908‐914.
   Glazov, E.A., Zankl, A., Donskoi, M., Kenna, T.J., Thomas, G.P., Clark, G.R., Duncan, E.L., and Brown, M.A. 2011. Whole‐exome re‐sequencing in a family quartet identifies POP1 mutations as the cause of a novel skeletal dysplasia. PLoS Genet. 7:e1002027.
   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.
   Hedges, D.J., Burges, D., Powell, E., Almonte, C., Huang, J., Young, S., Boese, B., Schmidt, M., Pericak‐Vance, M.A., Martin, E., Zhang, X., Harkins, T.T., and Züchner, S. 2009. Exome sequencing of a multigenerational human pedigree. PLoS One 4:e8232.
   Kalay, E., Yigit, G., Aslan, Y., Brown, K.E., Pohl, E., Bicknell, L.S., Kayserili, H., Li, Y., Tüysüz, B., Nürnberg, G., Kiess, W., Koegl, M., Baessmann, I., Buruk, K., Toraman, B., Kayipmaz, S., Kul, S., Ikbal, M., Turner, D.J., Taylor, M.S., Aerts, J., Scott, C., Milstein, K., Dollfus, H., Wieczorek, D., Brunner, H.G., Hurles, M., Jackson, A.P., Rauch, A., Nürnberg, P., Karagüzel, A., and Wollnik, B. 2011. CEP152 is a genome maintenance protein disrupted in Seckel syndrome. Nat. Genet. 43:23‐26.
   Lee, H., O'Connor, B.D., Merriman, B., Funari, V.A., Homer, N., Chen, Z., Cohn, D.H., and Nelson, S.F. 2009. Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing. BMC Genomics 10:646.
   Nazarian, R., Shi, H., Wang, Q., Kong, X., Koya, R.C., Lee, H., Chen, Z., Lee, M.K., Attar, N., Sazegar, H., Chodon, T., Nelson, S.F., McArthur, G., Sosman, J.A., Ribas, A., and Lo, R.S. 2010. Melanomas acquire resistance to B‐RAF(V600E) inhibition by RTK or N‐RAS upregulation. Nature 468:973‐977.
   Ng, S.B., Turner, E.H., Robertson, P.D., Flygare, S.D., Bigham, A.W., Lee, C., Shaffer, T., Wong, M., Bhattacharjee, A., Eichler, E.E., Bamshad, M., Nickerson, D.A., and Shendure, J. 2009. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272‐276.
   Ng, S.B., Bigham, A.W., Buckingham, K.J., Hannibal, M.C., McMillin, M.J., Gildersleeve, H.I., Beck, A.E., Tabor, H.K., Cooper, G.M., Mefford, H.C., Lee, C., Turner, E.H., Smith, J.D., Rieder, M.J., Yoshiura, K., Matsumoto, N., Ohta, T., Niikawa, N., Nickerson, D.A., Bamshad, M.J., and Shendure, J. 2010a. Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat. Genet. 42:790‐793.
   Ng, S.B., Buckingham, K.J., Lee, C., Bigham, A.W., Tabor, H.K., Dent, K.M., Huff, C.D., Shannon, P.T., Jabs, E.W., Nickerson, D.A., Shendure, J., and Bamshad, M.J. 2010b. Exome sequencing identifies the cause of a mendelian disorder. Nat. Genet. 42:30‐35.
   Shi, Y., Li, Y., Zhang, D., Zhang, H., Li, Y., Lu, F., Liu, X., He, F., Gong, B., Cai, L., Li, R., Liao, S., Ma, S., Lin, H., Cheng, J., Zheng, H., Shan, Y., Chen, B., Hu, J., Jin, X., Zhao, P., Chen, Y., Zhang, Y., Lin, Y., Li, X., Fan, Y., Yang, H., Wang, J., and Yang, Z. 2011. Exome sequencing identifies ZNF644 mutations in high myopia. PLoS Genet. 7:e1002084.
   Snape, K., Hanks, S., Ruark, E., Barros‐Núñez, P., Elliott, A., Murray, A., Lane, A.H., Shannon, N., Callier, P., Chitayat, D., Clayton‐Smith, J., Fitzpatrick, D.R., Gisselsson, D., Jacquemont, S., Asakura‐Hay, K., Micale, M.A., Tolmie, J., Turnpenny, P.D., Wright, M., Douglas, J., and Rahman, N. 2011. Mutations in CEP57 cause mosaic variegated aneuploidy syndrome. Nat. Genet. 43:527‐529.
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Key References
  Agilent Technologies. October 2009. SureSelect Human All Exon Kit, Illumina Paired‐End Sequencing Library Prep Protocol, Version 1.0.1.
  The product manuals for each of the described methods are included with the assay kits when ordered directly from the vendors. The protocols in this unit are derived from these manuals with some modifications.
  Nimblegen. December 2010. SeqCap EZ Exome Library SR User's Guide Version 2.2.
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