Abstract
Whole exome sequencing (WES) is a DNA sequencing strategy that provides a survey of base substitutions across coding genomic locations and other regions of interest. As the coding portion of the genome encompasses only 1–2% of the entire genome, this approach represents a more cost-effective strategy to detect DNA alterations that may alter protein function, compared to whole genome sequencing. Although the research community has and is currently delineating the functional implications of sequence changes in noncoding regions of the genome, WES is a currently available assay that provides valuable information for both discovery research and precision medicine applications. In this chapter, we present a WES library preparation protocol using the KAPA Hyper Prep Kit with Agilent SureSelect Human All Exon V5+UTR probes that demonstrates high DNA-to-library conversion efficiency for sequencing on the Illumina HiSeq platform.
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References
Ley TJ, Minx PJ, Walter MJ, Ries RE, Sun H, McLellan M, DiPersio JF, Link DC, Tomasson MH, Graubert TA et al (2003) A pilot study of high-throughput, sequence-based mutational profiling of primary human acute myeloid leukemia cell genomes. Proc Natl Acad Sci U S A 100:14275–14280. Epub 12003 Nov 14212
Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, Nayir A, Bakkaloglu A, Ozen S, Sanjad S et al (2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci U S A 106:19096–19101. 10.11073/pnas.0910672106. Epub 0910672009 Oct 0910672127
Musunuru K, Pirruccello JP, Do R, Peloso GM, Guiducci C, Sougnez C, Garimella KV, Fisher S, Abreu J, Barry AJ et al (2010) Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl J Med 363:2220–2227. https://doi.org/10.1056/NEJMoa1002926. Epub 1002010 Oct 1002913
Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, Huff CD, Shannon PT, Jabs EW, Nickerson DA et al (2010) Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 42:30–35. https://doi.org/10.1038/ng.1499. Epub 2009 Nov 1013
Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T, Wong M, Bhattacharjee A, Eichler EE et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272–276. https://doi.org/10.1038/nature08250. Epub 02009 Aug 08216
Kanchi KL, Johnson KJ, Lu C, McLellan MD, Leiserson MD, Wendl MC, Zhang Q, Koboldt DC, Xie M, Kandoth C et al (2014) Integrated analysis of germline and somatic variants in ovarian cancer. Nat Commun 5:3156. https://doi.org/10.1038/ncomms4156
Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, Nickerson E, Auclair D, Li L, Place C et al (2012) A landscape of driver mutations in melanoma. Cell 150:251–263. https://doi.org/10.1016/j.cell.2012.1006.1024
Krauthammer M, Kong Y, Ha BH, Evans P, Bacchiocchi A, McCusker JP, Cheng E, Davis MJ, Goh G, Choi M et al (2012) Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat Genet 44:1006–1014. https://doi.org/10.1038/ng.2359. Epub 2012 Jul 1029
The Cancer Genome Atlas Network (2012) Comprehensive molecular portraits of human breast tumours. Nature 490:61–70. https://doi.org/10.1038/nature11412. Epub 12012 Sep 11423
Barbieri CE, Baca SC, Lawrence MS, Demichelis F, Blattner M, Theurillat JP, White TA, Stojanov P, Van Allen E, Stransky N et al (2012) Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nat Genet 44:685–689. https://doi.org/10.1038/ng.2279
Wang L, Tsutsumi S, Kawaguchi T, Nagasaki K, Tatsuno K, Yamamoto S, Sang F, Sonoda K, Sugawara M, Saiura A et al (2012) Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency. Genome Res 22:208–219. https://doi.org/10.1101/gr.123109.123111. Epub 122011 Dec 123107
Rabbani B, Tekin M, Mahdieh N (2014) The promise of whole-exome sequencing in medical genetics. J Hum Genet 59:5–15. https://doi.org/10.1038/jhg.2013.1114. Epub 2013 Nov 1037
Knierim E, Lucke B, Schwarz JM, Schuelke M, Seelow D (2011) Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing. PLoS One 6:e28240. 10.21371/journal.pone.0028240. Epub 0022011 Nov 0028230
Poptsova MS, Il'icheva IA, Nechipurenko DY, Panchenko LA, Khodikov MV, Oparina NY, Polozov RV, Nechipurenko YD, Grokhovsky SL (2014) Non-random DNA fragmentation in next-generation sequencing. Sci Rep 4:4532. https://doi.org/10.1038/srep04532
Kivioja T, Vaharautio A, Karlsson K, Bonke M, Enge M, Linnarsson S, Taipale J (2011) Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods 9:72–74. https://doi.org/10.1038/nmeth.1778
Islam S, Zeisel A, Joost S, La Manno G, Zajac P, Kasper M, Lonnerberg P, Linnarsson S (2014) Quantitative single-cell RNA-seq with unique molecular identifiers. Nat Methods 11:163–166. https://doi.org/10.1038/nmeth.2772. Epub 2013 Dec 1022
Kou R, Lam H, Duan H, Ye L, Jongkam N, Chen W, Zhang S, Li S (2016) Benefits and challenges with applying unique molecular identifiers in next generation sequencing to detect low frequency mutations. PLoS One 11:e0146638. https://doi.org/10.1371/journal.pone.0146638. eCollection 0142016
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Liang, W.S. et al. (2018). Whole Exome Library Construction for Next Generation Sequencing. In: DiStefano, J. (eds) Disease Gene Identification. Methods in Molecular Biology, vol 1706. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7471-9_9
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DOI: https://doi.org/10.1007/978-1-4939-7471-9_9
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