Whole‑genome resequencing of 100 healthy individuals using DNA pooling

Wang,Xiaobin; Sui,Weiguo; Wu,Weiqing; Hou,Xianliang; Ou,Minglin; Xiang,Yueying; Dai,Yong

doi:10.3892/etm.2016.3797

November-2016 Volume 12 Issue 5

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November-2016 Volume 12 Issue 5

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Article Open Access

Whole‑genome resequencing of 100 healthy individuals using DNA pooling

Authors:
- Xiaobin Wang
- Weiguo Sui
- Weiqing Wu
- Xianliang Hou
- Minglin Ou
- Yueying Xiang
- Yong Dai
View Affiliations / Copyright

Affiliations: Health Management Centre, The Affiliated Guilin Hospital, Southern Medical University, Guilin, Guangxi 541000, P.R. China, Guangxi Key Laboratory of Metabolic Diseases Research, Guilin, Guangxi 541000, P.R. China, Health Management Centre, The Second Clinical Medical College, Jinan University, Shenzhen, Guangdong 518001, P.R. China

Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Pages: 3143-3150
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Published online on: October 11, 2016

https://doi.org/10.3892/etm.2016.3797
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Abstract

With the advent of next-generation sequencing technology, the cost of sequencing has significantly decreased. However, sequencing costs remain high for large‑scale studies. In the present study, DNA pooling was applied as a cost‑effective strategy for sequencing. The sequencing results for 100 healthy individuals obtained via whole‑genome resequencing and using DNA pooling are presented in the present study. In order to minimise the likelihood of systematic bias in sampling, paired‑end libraries with an insert size of 500 bp were prepared for all samples and then subjected to whole‑genome sequencing using four lanes for each library and resulting in at least a 30‑fold haploid coverage for each sample. The NCBI human genome build37 (hg19) was used as a reference genome for the present study and the short reads were aligned to the reference genome achieving 99.84% coverage. In addition, the average sequencing depth was 32.76. In total, ~3 million single‑nucleotide polymorphisms were identified, of which 99.88% were in the NCBI dbSNP database. Furthermore, ~600,000 small insertion/deletions, 500,000 structure variants, 5,000 copy number variations and 13,000 single nucleotide variants were identified. According to the present study, the whole genome has been sequenced for a small sample subjects from southern China for the first time. Furthermore, new variation sites were identified by comparing with the reference sequence, and new knowledge of the human genome variation was added to the human genomic databases. Furthermore, the particular distribution regions of variation were illustrated by analyzing various sites of variation, such as single‑nucleotide polymorphisms.

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1	International Human Genome Sequencing Consortium, . Finishing the euchromatic sequence of the human genome. Nature. 431:931–945. 2004. View Article : Google Scholar : PubMed/NCBI
2	Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray SJ, Greenman CD, Varela I, Lin ML, Ordóñez GR, Bignell GR, et al: A comprehensive catalogue of somatic mutations from a human cancer genome. Nature. 463:191–196. 2010. View Article : Google Scholar : PubMed/NCBI
3	Ley TJ, Mardis ER, Ding L, Fulton B, McLellan MD, Chen K, Dooling D, Dunford-Shore BH, McGrath S, Hickenbotham M, et al: DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature. 456:66–72. 2008. View Article : Google Scholar : PubMed/NCBI
4	Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T, Wong M, Bhattacharjee A, Eichler EE, Bamshad M, Nickerson DA and Shendure J: Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 461:272–276. 2009. View Article : Google Scholar : PubMed/NCBI
5	Maher CA, Kumar-Sinha C, Cao X, Kalyana-Sundaram S, Han B, Jing X, Sam L, Barrette T, Palanisamy N and Chinnaiyan AM: Transcriptome sequencing to detect gene fusions in cancer. Nature. 458:97–101. 2009. View Article : Google Scholar : PubMed/NCBI
6	Levy S, Sutton G, Ng PC, Feuk L, Halpern AL, Walenz BP, Axelrod N, Huang J, Kirkness EF, Denisov G, et al: The diploid genome sequence of an individual human. PLoS Biol. 5:e2542007. View Article : Google Scholar : PubMed/NCBI
7	Wheeler DA, Srinivasan M, Egholm M, Shen Y, Chen L, McGuire A, He W, Chen YJ, Makhijani V, Roth GT, et al: The complete genome of an individual by massively parallel DNA sequencing. Nature. 452:872–876. 2008. View Article : Google Scholar : PubMed/NCBI
8	Wang J, Wang W, Li R, Li Y, Tian G, Goodman L, Fan W, Zhang J, Li J, Zhang J, et al: The diploid genome sequence of an Asian individual. Nature. 456:60–65. 2008. View Article : Google Scholar : PubMed/NCBI
9	Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, et al: Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 456:53–59. 2008. View Article : Google Scholar : PubMed/NCBI
10	Kim JI, Ju YS, Park H, Kim S, Lee S, Yi JH, Mudge J, Miller NA, Hong D, Bell CJ, et al: A highly annotated whole-genome sequence of a Korean individual. Nature. 460:1011–1015. 2009.PubMed/NCBI
11	Shen H, Li J, Zhang J, Xu C, Jiang Y, Wu Z, Zhao F, Liao L, Chen J, Lin Y, et al: Comprehensive characterization of human genome variation by high coverage whole-genome sequencing of forty four Caucasians. PLoS One. 8:e594942013. View Article : Google Scholar : PubMed/NCBI
12	1000 Genomes Project Consortium; Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, Kang HM, Marth GT and McVean GA: An integrated map of genetic variation from 1,092 human genomes. Nature. 491:56–65. 2012. View Article : Google Scholar : PubMed/NCBI
13	Sham P, Bader JS, Craig I, O'Donovan M and Owen M: DNA Pooling: A tool for large-scale association studies. Nat Rev Genet. 3:862–871. 2002. View Article : Google Scholar : PubMed/NCBI
14	Bansal V: A statistical method for the detection of variants from next-generation resequencing of DNA pools. Bioinformatics. 26:i318–i324. 2010. View Article : Google Scholar : PubMed/NCBI
15	Bansal V, Tewhey R, Leproust EM and Schork NJ: Efficient and cost effective population resequencing by pooling and in-solution hybridization. PLoS One. 30:e183532011. View Article : Google Scholar
16	Carlson CS, Eberle MA, Rieder MJ, Smith JD, Kruglyak L and Nickerson DA: Additional SNPs and linkage-disequilibrium analyses are necessary for whole-genome association studies in humans. Nat Genet. 33:518–521. 2003. View Article : Google Scholar : PubMed/NCBI
17	Van Tassell CP, Smith TP, Matukumalli LK, Taylor JF, Schnabel RD, Lawley CT, Haudenschild CD, Moore SS, Warren WC and Sonstegard TS: SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nat Methods. 5:247–252. 2008. View Article : Google Scholar : PubMed/NCBI
18	Krumbiegel M, Pasutto F, Schlötzer-Schrehardt U, Uebe S, Zenkel M, Mardin CY, Weisschuh N, Paoli D, Gramer E, Becker C, et al: Genome-wide association study with DNA pooling identifies variants at CNTNAP2 associated with pseudoexfoliation syndrome. Eur J Hum Genet. 19:186–193. 2011. View Article : Google Scholar : PubMed/NCBI
19	Xiong Q, Ancona N, Hauser ER, Mukherjee S and Furey TS: Integrating genetic and gene expression evidence into genome-wide association analysis of gene sets. Genome Res. 22:386–397. 2012. View Article : Google Scholar : PubMed/NCBI
20	Ju YS, Kim JI, Kim S, Hong D, Park H, Shin JY, Lee S, Lee WC, Kim S, Yu SB, et al: Extensive genomic and transcriptional diversity identified through massively parallel DNA and RNA sequencing of eighteen Korean individuals. Nat Genet. 43:745–752. 2011. View Article : Google Scholar : PubMed/NCBI
21	Hantz S, Goudard M, Marczuk V, Renaudie J, Dussartre C, Bakeland D, Denis F and Alain S: HPV detection and typing by INNO-LiPA assay on liquid cytology media Easyfix Labonord after extraction QIAamp DNA Blood Mini Kit Qiagen and Nuclisens easyMAG Biomérieux. Pathol Biol (Paris). 58:179–183. 2010. View Article : Google Scholar : PubMed/NCBI
22	Villagomez R, Hatti-Kaul R, Sterner O, Almanza G and Linares-Pastén JA: Effect of natural and semisynthetic pseudoguianolides on the stability of NF-κB: DNA complex studied by agarose gel electrophoresis. PLoS One. 10:e01158192015. View Article : Google Scholar : PubMed/NCBI
23	Zhu N and Wang Z: An assay for DNA fragmentation in apoptosis without phenol/chloroform extraction and ethanol precipitation. Anal Biochem. 246:155–158. 1997. View Article : Google Scholar : PubMed/NCBI
24	Sambrook J and Russell DW: Fragmentation of DNA by nebulization. CSH Protoc. 4:45392006.
25	Chuchana P: Polymerase chain reaction: General methodology. Ann Biol Clin (Paris). 50:703–708. 1992.PubMed/NCBI
26	Mardis ER: Next-generation DNA sequencing platforms. Annu Rev Anal Chem (Palo Alto Calif). 6:287–303. 2013. View Article : Google Scholar : PubMed/NCBI
27	Li H and Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 26:589–595. 2010. View Article : Google Scholar : PubMed/NCBI
28	Li R, Li Y, Fang X, Yang H and Wang J, Kristiansen K and Wang J: SNP detection for massively parallel whole-genome resequencing. Genome Res. 19:1124–1132. 2009. View Article : Google Scholar : PubMed/NCBI
29	Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G and Durbin R: 1000 Genome Project Data Processing Subgroup: The sequence alignment/Map format and SAMtools. Bioinformatics. 25:2078–2079. 2009. View Article : Google Scholar : PubMed/NCBI
30	Koboldt DC, Chen K, Wylie T, Larson DE, McLellan MD, Mardis ER, Weinstock GM, Wilson RK and Ding L: VarScan: Variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics. 25:2283–2285. 2009. View Article : Google Scholar : PubMed/NCBI
31	Chen K, Wallis JW, McLellan MD, Larson DE, Kalicki JM, Pohl CS, McGrath SD, Wendl MC, Zhang Q, Locke DP, et al: BreakDancer: An algorithm for high-resolution mapping of genomic structural variation. Nat Methods. 6:677–681. 2009. View Article : Google Scholar : PubMed/NCBI
32	Chiang DY, Getz G, Jaffe DB, O'Kelly MJ, Zhao X, Carter SL, Russ C, Nusbaum C, Meyerson M and Lander ES: High-resolution mapping of copy-number alterations with massively parallel sequencing. Nat Methods. 6:99–103. 2009. View Article : Google Scholar : PubMed/NCBI
33	Wang K, Li M and Hakonarson H: ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acid Res. 38:e1642010. View Article : Google Scholar : PubMed/NCBI
34	Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi Y, Scherer SW and Lee C: Detection of large-scale variation in the human genome. Nat Genet. 36:949–951. 2004. View Article : Google Scholar : PubMed/NCBI
35	Out AA, van Minderhout IJ, Goeman JJ, Ariyurek Y, Ossowski S, Schneeberger K, Weigel D, van Galen M, Taschner PE, Tops CM, et al: Deep sequencing to reveal new variants in pooled DNA samples. Hum Mutat. 30:1703–1712. 2009. View Article : Google Scholar : PubMed/NCBI
36	Pleasance ED, Stephens PJ, O'Meara S, McBride DJ, Meynert A, Jones D, Lin ML, Beare D, Lau KW, Greenman C, et al: A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature. 463:184–190. 2010. View Article : Google Scholar : PubMed/NCBI
37	Meuwissen T and Goddard M: Accurate prediction of genetic values for complex traits by whole-genome resequencing. Genetics. 185:623–631. 2010. View Article : Google Scholar : PubMed/NCBI
38	Ingman M and Gyllensten U: SNP frequency estimation using massively parallel sequencing of pooled DNA. Eur J Hum Genet. 17:383–386. 2009. View Article : Google Scholar : PubMed/NCBI