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Methods of Gene Enrichment and Massively Parallel Sequencing Technologies

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Next Generation Sequencing

Abstract

Thirty years after the invention of dideoxy sequencing (a.k.a. Sanger sequencing), the advent of massively parallel sequencing technologies became another biotechnical revolution that enables the acquisition of genetic information in gigabase scale within an acceptable period of time. As a consequence, causal mutations underlying clinically heterogeneous disorders are more efficiently detected, paving the way for deciphering the pathogenecities of complex diseases. With the huge potential impact in modern medicine and health care, progress has been rapid in further optimizing the technology in both the academic and industrial fields. Third-generation sequencing technologies, although still facing multiple challenges, have shed light on the direct analyses of DNA and RNA at single-molecule level. Currently, before whole genome sequencing becomes routine, an in-depth assessment of targeted genomic regions is more feasible and has been widely applied in both clinical applications and basic research. Various enrichment methods, either PCR-based or hybridization-based, have been developed and gradually improved during application. This chapter provides detailed information on various target gene enrichment methods as well as massively parallel sequencing platforms. Hopefully this could assist the project-based approach/platform selections tailored for individual needs.

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References

  1. Margulies M, Egholm M, Altman WE et al (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437(7057):376–380

    PubMed  CAS  Google Scholar 

  2. Bilguvar K, Ozturk AK, Louvi A et al (2010) Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations. Nature 467(7312):207–210

    Article  PubMed  CAS  Google Scholar 

  3. Campeau PM, Lu JT, Sule G et al (2012) Whole-exome sequencing identifies mutations in the nucleoside transporter gene SLC29A3 in dysosteosclerosis, a form of osteopetrosis. Hum Mol Genet 21(22):4904–4909

    Article  PubMed  CAS  Google Scholar 

  4. O'Roak BJ, Deriziotis P, Lee C et al (2011) Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet 43(6):585–589

    Article  PubMed  Google Scholar 

  5. 1000 Genomes Project Consortium, Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA (2010) A map of human genome variation from population-scale sequencing. Nature 467(7319):1061–1073

    Article  PubMed  Google Scholar 

  6. Jones MA, Bhide S, Chin E et al (2011) Targeted polymerase chain reaction-based enrichment and next generation sequencing for diagnostic testing of congenital disorders of glycosylation. Genet Med 13(11):921–932

    Article  PubMed  CAS  Google Scholar 

  7. Wang J, Cui H, Lee N-C et al (2012) Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin. Genet Med 15(2):106–14

    Article  PubMed  Google Scholar 

  8. Gowrisankar S, Lerner-Ellis JP, Cox S et al (2010) Evaluation of second-generation sequencing of 19 dilated cardiomyopathy genes for clinical applications. J Mol Diagn 12(6):818–827

    Article  PubMed  CAS  Google Scholar 

  9. Baetens M, Van Laer L, De Leeneer K et al (2011) Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes. Hum Mutat 32(9):1053–1062

    Article  CAS  Google Scholar 

  10. Meuzelaar LS, Lancaster O, Pasche JP, Kopal G, Brookes AJ (2007) MegaPlex PCR: a strategy for multiplex amplification. Nat Methods 4(10):835–837

    Article  PubMed  CAS  Google Scholar 

  11. Tewhey R, Warner JB, Nakano M et al (2009) Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27(11):1025–1031

    Article  PubMed  CAS  Google Scholar 

  12. Kirkness EF (2009) Targeted sequencing with microfluidics. Nat Biotechnol 27(11):998–999

    Article  PubMed  CAS  Google Scholar 

  13. Schlipf NA, Schüle R, Klimpe S et al (2011) Amplicon-based high-throughput pooled sequencing identifies mutations in CYP7B1 and SPG7 in sporadic spastic paraplegia patients. Clin Genet 80(2):148–160

    Article  PubMed  CAS  Google Scholar 

  14. Ghadessy FJ, Ong JL, Holliger P (2001) Directed evolution of polymerase function by 0 self-replication. Proc Natl Acad Sci 98(8):4552–4557

    Article  PubMed  CAS  Google Scholar 

  15. Williams R, Peisajovich SG, Miller OJ, Magdassi S, Tawfik DS, Griffiths AD (2006) Amplification of complex gene libraries by emulsion PCR. Nat Methods 3(7):545–550

    Article  PubMed  CAS  Google Scholar 

  16. Valencia CA, Rhodenizer D, Bhide S et al (2012) Assessment of target enrichment platforms using massively parallel sequencing for the mutation detection for congenital muscular dystrophy. J Mol Diagn 14(3):233–246

    Article  PubMed  CAS  Google Scholar 

  17. Wheeler DA, Srinivasan M, Egholm M et al (2008) The complete genome of an individual by massively parallel DNA sequencing. Nature 452(7189):872–876

    Article  PubMed  CAS  Google Scholar 

  18. Levy S, Sutton G, Ng PC et al (2007) The diploid genome sequence of an individual human. PLoS Biol 5(10):e254

    Article  PubMed  Google Scholar 

  19. Zhang MQ (1998) Statistical features of human exons and their flanking regions. Hum Mol Genet 7(5):919–932

    Article  PubMed  CAS  Google Scholar 

  20. Sakharkar MK, Chow VT, Kangueane P (2004) Distributions of exons and introns in the human genome. In Silico Biol 4(4):387–393

    PubMed  CAS  Google Scholar 

  21. Barnes WM (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci USA 91(6):2216–2220

    Article  PubMed  CAS  Google Scholar 

  22. Tang S, Huang T (2010) Characterization of mitochondrial DNA heteroplasmy using a parallel sequencing system. Biotechniques 48(4):287–296

    Article  PubMed  CAS  Google Scholar 

  23. He Y, Wu J, Dressman DC et al (2010) Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Nature 464(7288):610–614

    Article  PubMed  CAS  Google Scholar 

  24. Zhang W, Cui H, Wong LJ (2012) Comprehensive 1-step molecular analyses of mitochondrial genome by massively parallel sequencing. Clin Chem 58(9):1322–1331

    Article  PubMed  CAS  Google Scholar 

  25. Li M, Schönberg A, Schaefer M, Schroeder R, Nasidze I, Stoneking M (2010) Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes. Am J Hum Genet 87(2):237–249

    Article  PubMed  CAS  Google Scholar 

  26. Cui H, Li F, Chen D, et al (2013) Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders. Genet Med. 2013 Jan 3. [Epub ahead of print], PMID 23288206

    Google Scholar 

  27. Coppieters F, De Wilde B, Lefever S et al (2012) Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis. Genet Med 14(6):576–585

    Article  PubMed  CAS  Google Scholar 

  28. Turner EH, Lee C, Ng SB, Nickerson DA, Shendure J (2009) Massively parallel exon capture and library-free resequencing across 16 genomes. Nat Methods 6(5):315–316

    Article  PubMed  CAS  Google Scholar 

  29. Dahl F, Stenberg J, Fredriksson S et al (2007) Multigene amplification and massively parallel sequencing for cancer mutation discovery. Proc Natl Acad Sci 104(22):9387–9392

    Article  PubMed  CAS  Google Scholar 

  30. Dahl F, Gullberg M, Stenberg J, Landegren U, Nilsson M (2005) Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments. Nucleic Acids Res 33(8):e71

    Article  PubMed  Google Scholar 

  31. Stenberg J, Dahl F, Landegren U, Nilsson M (2005) PieceMaker: selection of DNA fragments for selector-guided multiplex amplification. Nucleic Acids Res 33(8):e72

    Article  PubMed  Google Scholar 

  32. Fredriksson S, Banér J, Dahl F et al (2007) Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector. Nucleic Acids Res 35(7):e47

    Article  PubMed  Google Scholar 

  33. Porreca GJ, Zhang K, Li JB et al (2007) Multiplex amplification of large sets of human exons. Nat Methods 4(11):931–936

    Article  PubMed  CAS  Google Scholar 

  34. Hodges E, Xuan Z, Balija V et al (2007) Genome-wide in situ exon capture for selective resequencing. Nat Genet 39(12):1522–1527

    Article  PubMed  CAS  Google Scholar 

  35. Okou DT, Steinberg KM, Middle C, Cutler DJ, Albert TJ, Zwick ME (2007) Microarray-based genomic selection for high-throughput resequencing. Nat Methods 4(11):907–909

    Article  PubMed  CAS  Google Scholar 

  36. Albert TJ, Molla MN, Muzny DM et al (2007) Direct selection of human genomic loci by microarray hybridization. Nat Methods 4(11):903–905

    Article  PubMed  CAS  Google Scholar 

  37. Gnirke A, Melnikov A, Maguire J et al (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 27(2):182–189

    Article  PubMed  CAS  Google Scholar 

  38. Cancer Genome Atlas Research Network (2011) Integrated genomic analyses of ovarian carcinoma. Nature 474(7353):609–615

    Article  Google Scholar 

  39. Boileau C, Guo D-C, Hanna N et al (2012) TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome. Nat Genet 44(8):916–921

    Article  PubMed  CAS  Google Scholar 

  40. Glycogen storage diseases (GSDs) are a group of inherited genetic defects of glycogen synthesis or catabolism. GSDs are categorized into 14 subtypes, based on the specific enzyme deficiency and disease phenotype. Common symptoms include hypoglycemia, hepatomegaly, developmental delay and muscle cramps. Based on major clinical presentation, GSDs can be divided into two sub-forms: muscle and liver. This comprehensive panel includes genes involved in both the muscle and liver forms of GSDs

    Google Scholar 

  41. Metabolic myopathies are genetic disorders of energy metabolism due to defects in the pathways of carbohydrate and fatty acid catabolism, and the subsequent energy production through mitochondrial oxidative phosphorylation. Mutations in genes involved in three major pathways of energy metabolism, including glycogenolysis, fatty acid oxidation, and mitochondrial oxidative phosphorylation are the main causes of metabolic myopathies. These groups of diseases are clinically heterogeneous with variable penetrance, severity and age of onset. The predominant clinical symptoms associated with metabolic myopathy include chronic muscle weakness, myoglobinuria, and/or acute and recurrent episodes of irreversible muscle dysfunction related to exercise intolerance. Patients with metabolic myopathy are usually diagnosed based on their clinical features, abnormal metabolites, and enzymatic deficiency. However, the biochemical and molecular analytical procedures are time-consuming, costly, and often not confirmatory. Definitive diagnosis is made through the identification of deleterious mutations in the causative gene. Early diagnosis of these conditions is important for prompt clinical management and improved outcome of the patients

    Google Scholar 

  42. Oh JD, Kling-Backhed H, Giannakis M et al (2006) The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain: evolution during disease progression. Proc Natl Acad Sci USA 103(26):9999–10004

    Article  PubMed  CAS  Google Scholar 

  43. Smith MG, Gianoulis TA, Pukatzki S et al (2007) New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. Genes Dev 21(5):601–614

    Article  PubMed  CAS  Google Scholar 

  44. Girard A, Sachidanandam R, Hannon GJ, Carmell MA (2006) A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442(7099):199–202

    PubMed  Google Scholar 

  45. Tarasov V, Jung P, Verdoodt B et al (2007) Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 6(13):1586–1593

    Article  PubMed  CAS  Google Scholar 

  46. Wicker T, Schlagenhauf E, Graner A, Close T, Keller B, Stein N (2006) 454 sequencing put to the test using the complex genome of barley. BMC Genomics 7(1):275

    Article  PubMed  Google Scholar 

  47. Rothberg JM, Hinz W, Rearick TM et al (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475(7356):348–352

    Article  PubMed  CAS  Google Scholar 

  48. Elliott AM, Radecki J, Moghis B, Li X, Kammesheidt A (2012) Rapid detection of the ACMG/ACOG-recommended 23 CFTR disease-causing mutations using ion torrent semiconductor sequencing. J Biomol Tech 23(1):24–30

    Article  PubMed  Google Scholar 

  49. Vogel U, Szczepanowski R, Claus H, Junemann S, Prior K, Harmsen D (2012) Ion torrent personal genome machine sequencing for genomic typing of Neisseria meningitidis for rapid determination of multiple layers of typing information. J Clin Microbiol 50(6):1889–1894

    Article  PubMed  CAS  Google Scholar 

  50. Whiteley AS, Jenkins S, Waite I et al (2012) Microbial 16S rRNA Ion Tag and community metagenome sequencing using the Ion Torrent (PGM) Platform. J Microbiol Methods 91(1):80–88

    Article  PubMed  CAS  Google Scholar 

  51. Junemann S, Prior K, Szczepanowski R et al (2012) Bacterial community shift in treated periodontitis patients revealed by ion torrent 16S rRNA gene amplicon sequencing. PLoS One 7(8):e41606

    Article  PubMed  Google Scholar 

  52. Eid J, Fehr A, Gray J et al (2009) Real-time DNA sequencing from single polymerase molecules. Science 323(5910):133–138

    Article  PubMed  CAS  Google Scholar 

  53. Meissner A (2010) Epigenetic modifications in pluripotent and differentiated cells. Nat Biotechnol 28(10):1079–1088

    Article  PubMed  CAS  Google Scholar 

  54. Esteller M (2007) Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 8(4):286–298

    Article  PubMed  CAS  Google Scholar 

  55. Delaval K, Feil R (2004) Epigenetic regulation of mammalian genomic imprinting. Curr Opin Genet Dev 14(2):188–195

    Article  PubMed  CAS  Google Scholar 

  56. Dobrovic A, Simpfendorfer D (1997) Methylation of the BRCA1 gene in sporadic breast cancer. Cancer Res 57(16):3347–3350

    PubMed  CAS  Google Scholar 

  57. Plath K, Mlynarczyk-Evans S, Nusinow DA, Panning B (2002) Xist RNA and the mechanism of X chromosome inactivation. Annu Rev Genet 36:233–278

    Article  PubMed  CAS  Google Scholar 

  58. Horsthemke B, Wagstaff J (2008) Mechanisms of imprinting of the Prader–Willi/Angelman region. Am J Med Genet A 146A(16):2041–2052

    Article  PubMed  CAS  Google Scholar 

  59. Taiwo O, Wilson GA, Morris T et al (2012) Methylome analysis using MeDIP-seq with low DNA concentrations. Nat Protoc 7(4):617–636

    Article  PubMed  CAS  Google Scholar 

  60. Harris RA, Wang T, Coarfa C et al (2010) Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications. Nat Biotechnol 28(10):1097–1105

    Article  PubMed  CAS  Google Scholar 

  61. Serre D, Lee BH, Ting AH (2010) MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome. Nucleic Acids Res 38(2):391–399

    Article  PubMed  CAS  Google Scholar 

  62. Ball MP, Li JB, Gao Y et al (2009) Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nat Biotechnol 27(4):361–368

    Article  PubMed  CAS  Google Scholar 

  63. Flusberg BA, Webster DR, Lee JH et al (2010) Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7(6):461–465

    Article  PubMed  CAS  Google Scholar 

  64. Branton D, Deamer DW, Marziali A et al (2008) The potential and challenges of nanopore sequencing. Nat Biotechnol 26(10):1146–1153

    Article  PubMed  CAS  Google Scholar 

  65. Pennisi E (2012) 23 July, 2012. New start date and first contestant for genomics X PRIZE. ScienceInsider

    Google Scholar 

  66. von Bubnoff A (2008) Next-generation sequencing: the race is on. Cell 132(5):721–723

    Article  Google Scholar 

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Acknowledgments

I sincerely thank Drs. Jing Wang, Megan L. Landsverk, Victor W. Zhang, and Lee-Jun Wong for reviewing this manuscript and their invaluable comments.

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Authors and Affiliations

  1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA

    Hong Cui Ph.D.

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  1. Hong Cui Ph.D.
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Correspondence to Hong Cui Ph.D. .

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  1. , Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor Plaza, Houston, 77030, Texas, USA

    Lee-Jun C. Wong

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Cui, H. (2013). Methods of Gene Enrichment and Massively Parallel Sequencing Technologies. In: Wong, LJ. (eds) Next Generation Sequencing. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-7001-4_3

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