Exome sequencing: the sweet spot before whole genomes

Jamie K Teer, James C Mullikin, Jamie K Teer, James C Mullikin

Abstract

The development of massively parallel sequencing technologies, coupled with new massively parallel DNA enrichment technologies (genomic capture), has allowed the sequencing of targeted regions of the human genome in rapidly increasing numbers of samples. Genomic capture can target specific areas in the genome, including genes of interest and linkage regions, but this limits the study to what is already known. Exome capture allows an unbiased investigation of the complete protein-coding regions in the genome. Researchers can use exome capture to focus on a critical part of the human genome, allowing larger numbers of samples than are currently practical with whole-genome sequencing. In this review, we briefly describe some of the methodologies currently used for genomic and exome capture and highlight recent applications of this technology.

Figures

Figure 1.
Figure 1.
Illustration of different capture methods. Light blue bars represent desired genomic sequence, red bars represent unwanted sequence. (A) Solid-phase hybridization. Bait probes (light blue and black) complementary to the desired sequence are synthesized on a microarray. Fragmented genomic DNA is applied, and the desired fragments hybridize. The array is washed, and desired fragments are eluted. (B) Liquid-phase hybridization. Bait probes (light blue and black) complementary to the desired regions are synthesized, often using microarray technology. The probes are generally biotinylated (asterisk). The bait probes are mixed with fragmented genomic DNA, and the desired fragments hybridize to baits in solution. Streptavidin beads (black circles) are added to allow physical separation. The bead-bait complexes are washed, and desired DNA is eluted. (C) MIP. Single-stranded probes composed of a universal linker backbone (black line) and arms complementary to the sequence flanking desired regions (red and white) are synthesized, often using microarray or microfluidics technology. The probes are added to genomic DNA and hybridize in an inverted manner. A polymerase (yellow oval) fills in the gap between the two arms. A ligase (yellow star) seals the nick, resulting in a closed single-strand circle. Genomic DNA is digested with exonucleases, and the captured DNA is amplified using sequences in the universal backbone. (D) PEC. Biotinylated primers (red and white) are added to fragmented genomic DNA, where they hybridize to the desired sequence. A polymerase (yellow oval) extends the primer, creating a tighter interaction. Streptavidin beads (black circles) are added and are used to physically separate the desired DNA from the unwanted DNA. The desired DNA is then eluted.

References

    1. Tarpey P.S., Smith R., Pleasance E., Whibley A., Edkins S., Hardy C., O'Meara S., Latimer C., Dicks E., Menzies A., et al. A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. Nat. Genet. 2009;41:535–543. .
    1. Jones S., Zhang X., Parsons D.W., Lin J.C., Leary R.J., Angenendt P., Mankoo P., Carter H., Kamiyama H., Jimeno A., et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 2008;321:1801–1806. .
    1. Garber K. Fixing the front end. Nat. Biotechnol. 2008;26:1101–1104. .
    1. Summerer D. Enabling technologies of genomic-scale sequence enrichment for targeted high-throughput sequencing. Genomics. 2009;94:363–368. .
    1. Turner E.H., Ng S.B., Nickerson D.A., Shendure J. Methods for genomic partitioning. Annu. Rev. Genomics Hum. Genet. 2009;10:263–284. .
    1. Mamanova L., Coffey A.J., Scott C.E., Kozarewa I., Turner E.H., Kumar A., Howard E., Shendure J., Turner D.J. Target-enrichment strategies for next-generation sequencing. Nat. Methods. 2010;7:111–118. .
    1. 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., et al. Direct selection of human genomic loci by microarray hybridization. Nat. Methods. 2007;4:903–905. .
    1. Okou D.T., Steinberg K.M., Middle C., Cutler D.J., Albert T.J., Zwick M.E. Microarray-based genomic selection for high-throughput resequencing. Nat. Methods. 2007;4:907–909. .
    1. 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., et al. Genome-wide in situ exon capture for selective resequencing. Nat. Genet. 2007;39:1522–1527. .
    1. Hodges E., Rooks M., Xuan Z., Bhattacharjee A., Benjamin Gordon D., Brizuela L., Richard McCombie W., Hannon G.J. Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nat. Protoc. 2009;4:960–974. .
    1. Bau S., Schracke N., Kranzle M., Wu H., Stahler P.F., Hoheisel J.D., Beier M., Summerer D. Targeted next-generation sequencing by specific capture of multiple genomic loci using low-volume microfluidic DNA arrays. Anal. Bioanal. Chem. 2009;393:171–175. .
    1. Herman D.S., Hovingh G.K., Iartchouk O., Rehm H.L., Kucherlapati R., Seidman J.G., Seidman C.E. Filter-based hybridization capture of subgenomes enables resequencing and copy-number detection. Nat. Methods. 2009;6:507–510. .
    1. Summerer D., Wu H., Haase B., Cheng Y., Schracke N., Stahler C.F., Chee M.S., Stahler P.F., Beier M. Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing. Genome Res. 2009;19:1616–1621. .
    1. Lee H., O'Connor B.D., Merriman B., Funari V.A., Homer N., Chen Z., Cohn D.H., Nelson S.F. Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing. BMC Genomics. 2009;10:646. .
    1. Gnirke A., Melnikov A., Maguire J., Rogov P., LeProust E.M., Brockman W., Fennell T., Giannoukos G., Fisher S., Russ C., et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 2009;27:182–189. .
    1. 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., et al. Multiplex amplification of large sets of human exons. Nat. Methods. 2007;4:931–936. .
    1. Li J.B., Gao Y., Aach J., Zhang K., Kryukov G.V., Xie B., Ahlford A., Yoon J.K., Rosenbaum A.M., Zaranek A.W., et al. Multiplex padlock targeted sequencing reveals human hypermutable CpG variations. Genome Res. 2009;19:1606–1615. .
    1. Turner E.H., Lee C., Ng S.B., Nickerson D.A., Shendure J. Massively parallel exon capture and library-free resequencing across 16 genomes. Nat. Methods. 2009;6:315–316. .
    1. Deng J., Shoemaker R., Xie B., Gore A., LeProust E.M., Antosiewicz-Bourget J., Egli D., Maherali N., Park I.H., Yu J., et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat. Biotechnol. 2009;27:353–360. .
    1. Krishnakumar S., Zheng J., Wilhelmy J., Faham M., Mindrinos M., Davis R. A comprehensive assay for targeted multiplex amplification of human DNA sequences. Proc. Natl Acad. Sci. USA. 2008;105:9296–9301. .
    1. Briggs A.W., Good J.M., Green R.E., Krause J., Maricic T., Stenzel U., Lalueza-Fox C., Rudan P., Brajkovic D., Kucan Z., et al. Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science. 2009;325:318–321. .
    1. Tewhey R., Warner J.B., Nakano M., Libby B., Medkova M., David P.H., Kotsopoulos S.K., Samuels M.L., Hutchison J.B., Larson J.W., et al. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat. Biotechnol. 2009;27:1025–1031. .
    1. Ibrahim S.F., van den Engh G. High-speed chromosome sorting. Chromosome Res. 2004;12:5–14. .
    1. Weise A., Timmermann B., Grabherr M., Werber M., Heyn P., Kosyakova N., Liehr T., Neitzel H., Konrat K., Bommer C., et al. High-throughput sequencing of microdissected chromosomal regions. Eur. J. Hum. Genet. 2010;18:457–462. .
    1. Choi M., Scholl U.I., Ji W., Liu T., Tikhonova I.R., Zumbo P., Nayir A., Bakkaloglu A., Ozen S., Sanjad S., et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc. Natl Acad. Sci. USA. 2009;106:19096–19101. .
    1. 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., et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 2009;461:272–276. .
    1. 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., et al. Whole exome capture in solution with 3 Gbp of data. Genome Biol. 2010;11:R62. .
    1. Pruitt K.D., Harrow J., Harte R.A., Wallin C., Diekhans M., Maglott D.R., Searle S., Farrell C.M., Loveland J.E., Ruef B.J., et al. The consensus coding sequence (CCDS) project: Identifying a common protein-coding gene set for the human and mouse genomes. Genome Res. 2009;19:1316–1323. .
    1. Chou L.S., Liu C.S., Boese B., Zhang X., Mao R. DNA sequence capture and enrichment by microarray followed by next-generation sequencing for targeted resequencing: neurofibromatosis type 1 gene as a model. Clin. Chem. 2010;56:62–72. .
    1. Hoischen A., Gilissen C., Arts P., Wieskamp N., van der Vliet W., Vermeer S., Steehouwer M., de Vries P., Meijer R., Seiqueros J., et al. Massively parallel sequencing of ataxia genes after array-based enrichment. Hum. Mutat. 2010;31:494–499. .
    1. Raca G., Jackson C., Warman B., Bair T., Schimmenti L.A. Next generation sequencing in research and diagnostics of ocular birth defects. Mol. Genet. Metab., 2010;100:184–192.
    1. Brkanac Z., Spencer D., Shendure J., Robertson P.D., Matsushita M., Vu T., Bird T.D., Olson M.V., Raskind W.H. IFRD1 is a candidate gene for SMNA on chromosome 7q22–q23. Am. J. Hum. Genet. 2009;84:692–697. .
    1. Volpi L., Roversi G., Colombo E.A., Leijsten N., Concolino D., Calabria A., Mencarelli M.A., Fimiani M., Macciardi F., Pfundt R., et al. Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene. Am. J. Hum. Genet. 2010;86:72–76. .
    1. Nikopoulos K., Gilissen C., Hoischen A., van Nouhuys C.E., Boonstra F.N., Blokland E.A., Arts P., Wieskamp N., Strom T.M., Ayuso C., et al. Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy. Am. J. Hum. Genet. 2010;86:240–247. .
    1. Rehman A.U., Morell R.J., Belyantseva I.A., Khan S.Y., Boger E.T., Shahzad M., Ahmed Z.M., Riazuddin S., Khan S.N., Friedman T.B. Targeted capture and next-generation sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79. Am. J. Hum. Genet. 2010;86:378–388. .
    1. Johnston J.J., Teer J.K., Cherukuri P.F., Hansen N.F., Loftus S.K., Chong K., Mullikin J.C., Biesecker L.G. Massively parallel sequencing of exons on the X chromosome identifies RBM10 as the gene that causes a syndromic form of cleft palate. Am. J. Hum. Genet. 2010;86:743–748. .
    1. Burbano H.A., Hodges E., Green R.E., Briggs A.W., Krause J., Meyer M., Good J.M., Maricic T., Johnson P.L., Xuan Z., et al. Targeted investigation of the Neandertal genome by array-based sequence capture. Science. 2010;328:723–725. .
    1. Yi X., Liang Y., Huerta-Sanchez E., Jin X., Cuo Z.X.P., Pool J.E., Xu X., Jiang H., Vinckenbosch N., Korneliussen T.S., et al. Sequencing of 50 human exomes reveals adaptation to high altitude. Science. 2010;329:75–78. .
    1. Beall C.M., Cavalleri G.L., Deng L., Elston R.C., Gao Y., Knight J., Li C., Li J.C., Liang Y., McCormack M., et al. Natural selection on EPAS1 (HIF2alpha) associated with low hemoglobin concentration in Tibetan highlanders. Proc. Natl Acad. Sci. USA. 2010;107:11459–11464. .
    1. Simonson T.S., Yang Y., Huff C.D., Yun H., Qin G., Witherspoon D.J., Bai Z., Lorenzo F.R., Xing J., Jorde L.B., et al. Genetic evidence for high-altitude adaptation in Tibet. Science. 2010;329:72–75. .
    1. Hodges E., Smith A.D., Kendall J., Xuan Z., Ravi K., Rooks M., Zhang M.Q., Ye K., Bhattacharjee A., Brizuela L., et al. High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing. Genome Res. 2009;19:1593–1605. .
    1. Conrad D.F., Bird C., Blackburne B., Lindsay S., Mamanova L., Lee C., Turner D.J., Hurles M.E. Mutation spectrum revealed by breakpoint sequencing of human germline CNVs. Nat. Genet. 2010;42:385–391. .
    1. Li J.B., Levanon E.Y., Yoon J.K., Aach J., Xie B., Leproust E., Zhang K., Gao Y., Church G.M. Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing. Science. 2009;324:1210–1213. .
    1. Heap G.A., Yang J.H., Downes K., Healy B.C., Hunt K.A., Bockett N., Franke L., Dubois P.C., Mein C.A., Dobson R.J., et al. Genome-wide analysis of allelic expression imbalance in human primary cells by high-throughput transcriptome resequencing. Hum. Mol. Genet. 2010;19:122–134. .
    1. Levin J.Z., Berger M.F., Adiconis X., Rogov P., Melnikov A., Fennell T., Nusbaum C., Garraway L.A., Gnirke A. Targeted next-generation sequencing of a cancer transcriptome enhances detection of sequence variants and novel fusion transcripts. Genome Biol. 2009;10:R115. .

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