Skip to main content

Genome Sequencing

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1525))

Abstract

Genome sequencing is an important step toward correlating genotypes with phenotypic characters. Sequencing technologies are important in many fields in the life sciences, including functional genomics, transcriptomics, oncology, evolutionary biology, forensic sciences, and many more. The era of sequencing has been divided into three generations. First generation sequencing involved sequencing by synthesis (Sanger sequencing) and sequencing by cleavage (Maxam-Gilbert sequencing). Sanger sequencing led to the completion of various genome sequences (including human) and provided the foundation for development of other sequencing technologies. Since then, various techniques have been developed which can overcome some of the limitations of Sanger sequencing. These techniques are collectively known as “Next-generation sequencing” (NGS), and are further classified into second and third generation technologies. Although NGS methods have many advantages in terms of speed, cost, and parallelism, the accuracy and read length of Sanger sequencing is still superior and has confined the use of NGS mainly to resequencing genomes. Consequently, there is a continuing need to develop improved real time sequencing techniques. This chapter reviews some of the options currently available and provides a generic workflow for sequencing a genome.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   99.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   139.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Mardis EM (2011) A decade’s perspective on DNA sequencing technology. Nature 470:198–203F

    Article  CAS  PubMed  Google Scholar 

  2. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pareek CS, Smoczynski R, Tretyn A (2011) Sequencing technologies and genome sequencing. J Appl Genet 52:413–435

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM et al (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496–512

    Article  CAS  PubMed  Google Scholar 

  5. Venter JC, Adams MD, Myers EW et al (2001) The sequence of the human genome. Science 291:1304–1351

    Article  CAS  PubMed  Google Scholar 

  6. Koboldt DC, Steinberg KM, Larson DE, Wilson RK, Mardis EM (2013) The next generation sequencing revolution and its impact on genomics. Cell 155:27–38

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bormann Chung CA, Boyd VL, McKernan KJ, Fu YT, Monighetti C, Peckham HE, Barker M (2010) Whole methylome analysis by ultra-deep sequencing using two-base encoding. PLoS One 5:1–8

    Article  Google Scholar 

  8. Nowrousian M (2010) Next-generation sequencing techniques for eukaryotic microorganisms: sequencing-based solutions to biological problems. Eukaryot Cell 9:1300–1310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Koboldt DC, Larson DE, Chen K, Ding L, Wilson RK (2012) Massively parallel sequencing approaches for characterization of structural variation. Methods Mol Biol 838:369–384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Brautigam A, Gowik U (2010) What can next generation sequencing do for you? Next-generation sequencing as a valuable tool in plant research. Plant Biol 12:831–841

    Article  CAS  PubMed  Google Scholar 

  11. Thudi M, Li Y, Jackson SA, May GD, Varshney RK (2012) Current state-of-art sequencing technologies for plant genomics research. Brief Funct Genomics 2:3–11

    Article  Google Scholar 

  12. Pop M, Kosack D, Salzberg SL (2002) A hierarchical approach to building contig scaffolds. In: Second annual RECOMB satellite meeting on DNA sequencing and characterization. Stanford University

    Google Scholar 

  13. Shultz JL, Yesudas C, Yaegashi S, Afzal AJ, Kazi S, Lightfoot DA (2006) Three minimum tile paths from bacterial artificial chromosome libraries of soyabean (Glycine max cv Forrest): tools for structural and functional genomics. Plant Methods 2:9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L, Law M (2012) Comparison of next-generation sequencing systems. J Biomed Biotechnol 2012:1–11

    PubMed  Google Scholar 

  15. Edwards A, Caskey T (1991) Closure strategies for random DNA sequencing. Methods 3:41–47

    Article  CAS  Google Scholar 

  16. Chaisson MJ, Brinza D, Pevzner PA (2010) De novo fragment assembly with short mate-paired reads: does the read length matter? Genome Res 19:336–346

    Article  Google Scholar 

  17. Green P (1997) Against a whole-genome shotgun. Genome Res 7:410–417

    CAS  PubMed  Google Scholar 

  18. Stranneheim H, Lundeberg J (2012) Stepping stones in DNA sequencing. Biotechnol J 7:1063–1073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hutchison CA (2007) DNA sequencing: bench to bedside and beyond. Nucleic Acids Res 35:6227–6237

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Maxam MA, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci U S A 74(2):560–564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zimmermann J, Voss H, Schwager C, Stegemann J, Ansorge W (1989) Automated Sanger dideoxy sequencing reaction protocol. FEBS Lett 223:432–436

    Google Scholar 

  22. Ansorge W, Voss H, Wirkner U, Schwager C, Stegemann J, Pepperkok R, Zimmermann J, Erfle H (1989) Automated Sanger DNA sequencing with one label in less than four lanes on gel. J Biochem Biophys Methods 20:47–52

    Article  CAS  PubMed  Google Scholar 

  23. Rosenthal A, Charnock-Jones DS (1992) New protocols for DNA sequencing with dye terminators. DNA Seq 3:61–64

    Article  CAS  PubMed  Google Scholar 

  24. Franca LTC, Carrilho E, Kist TBL (2002) A review of DNA sequencing techniques. Q Rev Biophys 35:169–200

    Article  CAS  PubMed  Google Scholar 

  25. Bubnoff AV (2008) Next-generation sequencing: the race is on. Cell 132:721–723

    Article  Google Scholar 

  26. Metzker ML (2005) Emerging technologies in DNA sequencing. Genome Res 15:1767–1776

    Article  CAS  PubMed  Google Scholar 

  27. Shendure J, Ji H (2008) Next-generation DNA sequencing. Nat Biotechnol 26:1135–1145

    Article  CAS  PubMed  Google Scholar 

  28. Mardis EA (2013) Next-generation sequencing platforms. Annu Rev Anal Chem 6:287–303

    Article  CAS  Google Scholar 

  29. Blazej RG, Kumaresan P, Mathies RA (2006) Micro fabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. Proc Natl Acad Sci U S A 103:7240–7245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Augustin MA, Ankenbauer W, Angerer B (2001) Progress towards single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA. J Biotechnol 86:289–301

    Article  CAS  PubMed  Google Scholar 

  31. Hui P (2014) Next- generation sequencing: chemistry, technology and application. Top Curr Chem 336:1–18

    Article  CAS  PubMed  Google Scholar 

  32. Hert DG, Fredlake CP, Annelise E (2008) Advantages and limitations of next-generation sequencing technologies: a comparison of electrophoresis and non-electrophoresis methods. Electrophoresis 29:4618–4626

    Article  CAS  PubMed  Google Scholar 

  33. Mardis EA (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9:387–402

    Article  CAS  PubMed  Google Scholar 

  34. Ansorge WJ (2009) Next-generation sequencing techniques. New Biotechnol 25:195–203

    Article  CAS  Google Scholar 

  35. Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281:363–365

    Article  CAS  PubMed  Google Scholar 

  36. Keijser BJ, Zaura E, Huse SM, van der Vossen JM, Schuren FH, Montijn RC, ten Cate JM, Crielaard W (2008) Pyrosequencing analysis of the oral microflora of healthy adults. J Dent Res 87:1016–1020

    Article  CAS  PubMed  Google Scholar 

  37. Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR et al (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53–59

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Turcatti G, Romieu A, Fedurco M, Tairi AP (2008) A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis. Nucleic Acids Res 36:1–13

    Article  Google Scholar 

  39. Pettersson E, Lundeberg J, Ahmadian A (2009) Generations of sequencing technologies. Genomics 93:105–111

    Article  CAS  PubMed  Google Scholar 

  40. Niedringhaus TP, Milanova D, Kerby MB, Snyder MP, Barron AE (2011) Landscape of next-generation sequencing technologies. Anal Chem 83:4327–4341

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Voelkerding KV, Dames SA, Durtschi JD (2009) Next-generation sequencing: from basic research to diagnostic. Clin Chem 55(4):641–658

    Article  CAS  PubMed  Google Scholar 

  42. Metzker ML (2010) Sequencing technologies—next generation. Nat Rev Genet 11:31–46

    Article  CAS  PubMed  Google Scholar 

  43. Glenn TC (2011) Field guide to next-generation DNA sequencers. Mol Ecol Resour 11:759–769

    Article  CAS  PubMed  Google Scholar 

  44. Delsenya M, Han B, Hsing YI (2010) High throughput DNA sequencing: the new sequencing revolution. Plant Sci 179:407–422

    Article  Google Scholar 

  45. Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M et al (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475:348–352

    Article  CAS  PubMed  Google Scholar 

  46. Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Baybayan P, Bettman B et al (2010) Real-time DNA sequencing from single polymerase molecules. Science 323:133–138

    Article  Google Scholar 

  47. Kaji N, Okamoto Y, Tokeshi M, Baba Y (2010) Nanopillar, nanoball, and nanofibers for highly efficient analysis of biomolecules. Chem Soc Rev 39:948–956

    Article  CAS  PubMed  Google Scholar 

  48. Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, Carnevali P, Nazarenko I, Nilsen GB, Yeung G et al (2010) Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science 327:78–81

    Article  CAS  PubMed  Google Scholar 

  49. Porreca GJ (2010) Genome sequencing on nanoballs. Nat Biotechnol 28:43–44

    Article  CAS  PubMed  Google Scholar 

  50. Korlach J, Marks PJ, Cicero RL, Gray JJ, Murphy DL, Roitman DB, Pham TT, Otto GA, Foquet M, Turner SW (2008) Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. Proc Natl Acad Sci U S A 105:1176–1181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Korlach J, Bjornson KP, Chaudhuri BP, Cicero RL, Flusberg BA, Gray JJ, Holden D, Saxena R, Wegener J, Turner SW (2010) Real-time DNA sequencing from single polymerase molecules. Methods Enzymol 472:431–455

    Article  CAS  PubMed  Google Scholar 

  52. Schadt E, Turner S, Kasarskis A (2010) A window into third-generation sequencing. Hum Mol Genet 19(2):227–240

    Article  Google Scholar 

  53. Venkatesan BM, Bashir R (2011) Nanopore sensors for nucleic acid analysis. Nat Nanotechnol 6:615–624

    Article  CAS  PubMed  Google Scholar 

  54. Clarke J, Wu HC, Jayasinghe L, Patel A, Reid S, Bayley H (2009) Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol 4:265–270

    Article  CAS  PubMed  Google Scholar 

  55. Stoddart D, Heron AJ, Mikhailova E, Maglia G, Bayley H (2009) Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc Natl Acad Sci U S A 106:7702–7707

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Astier Y, Braha O, Bayley H (2006) Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5′-monophosphates by using an engineered protein nanopore equipped with a molecular adapter. J Am Chem Soc 128:1705–1710

    Article  CAS  PubMed  Google Scholar 

  57. Maitra RD, Kim J, Dunbar WB (2012) Recent advances in nanopore sequencing. Electrophoresis 33:3418–3428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Haque F, Li J, Wu HC, Liang XJ, Guo P (2013) Solid state and biological nanopore for real time sensing of single chemical and sequencing of DNA. Nano Today 8:56–74

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Lim JS, Choi BS, Lee JS, Shin C, Yang TJ, Rhee JS, Lee JS, Choi IY (2012) Survey of the applications of NGS to whole genome sequencing and expression profiling. Genomics Inform 10:1–8

    Article  PubMed  PubMed Central  Google Scholar 

  60. Thompson JF, Milos PM (2011) The properties and applications of single-molecule DNA sequencing. Genome Biol 12:217

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Zhou X, Ren L, Meng Q, Li Y, Yu Y, Yu J (2010) The next generation sequencing technology and application. Protein Cell 1:520–536

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Buermans HPJ, Dunnen JTD (2014) Next generation sequencing technology: advances and applications. Biochim Biophys Acta 1842:1932–1941

    Article  CAS  PubMed  Google Scholar 

  63. Warshauer DH, Lin D, Hari K, Jain R, Davis C, Larue B, King JL, Budowle B (2013) STRait Razor: a length-based forensic STR allele-calling tool for use with second generation sequencing data. Forensic Sci Int Genet 7(4):409–417

    Article  CAS  PubMed  Google Scholar 

  64. Kumar S, Banks TW, Cloutier S (2012) SNP discovery through next-generation sequencing and its applications. Int J Plant Genomics 2012:1–15

    Google Scholar 

  65. Berglund EC, Anna Kiialainen A, Syvänen AN (2011) Next generation sequencing technologies and applications for human genetic history and forensics. Investigative Genet 2:1–15

    Article  Google Scholar 

  66. Ozsolak F (2012) Third generation sequencing techniques and applications to drug discovery. Expert Opin Drug Discov 7:231–243

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Yadav NK, Shukla P, Omer A, Pareek S, Singh RK (2014) Next-generation sequencing: potential and application to drug discovery. Scientific World J 2014:1–7

    Google Scholar 

  68. Snyder M, Du J, Gerstein M (2010) Personal genome sequencing: current approaches and challenges. Genes Dev 23:423–431

    Article  Google Scholar 

  69. Yngvadottir B, MacArthur DG, Jin H, Tyler-Smith C (2009) The promise and reality of personal genomics. Genome Biol 10:237.1–237.4

    Article  Google Scholar 

  70. Grumbt B, Eck SH, Hinrichsen T, Hirv K (2013) Diagnostic applications of next generation sequencing in immunogenetics and molecular oncology. Transfus Med Hemother 40:196–206

    Article  PubMed  PubMed Central  Google Scholar 

  71. Haimovich AD (2011) Methods, challenges and promise of next generation sequencing in cancer biology. Yale J Biol Med 84:439–446

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Xuan J, Yu Y, Qing T, Guo L, Shi L (2013) Next-generation sequencing in the clinic: promises and challenges. Cancer Lett 340:284–295

    Article  CAS  PubMed  Google Scholar 

  73. Hall N (2007) Advanced sequencing technologies and their wider impact in microbiology. J Exp Biol 209:1518–1525

    Article  Google Scholar 

  74. Dijk ELV, Auger H, Jaszczyszyn Y, Thermes C (2014) Ten years of next-generation sequencing technology. Trends Genet 30(9):418–426

    Article  PubMed  Google Scholar 

  75. Morey M, Fernández-Marmiesse A, Castiñeiras D, Fraga JM, Couce ML, Cocho JA (2013) A glimpse into past, present, and future DNA sequencing. Mol Genet Metab 110:3–24

    Article  CAS  PubMed  Google Scholar 

  76. Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred: II. Error probabilities. Genome Res 8(3):186–194

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Mansi Verma .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Verma, M., Kulshrestha, S., Puri, A. (2017). Genome Sequencing. In: Keith, J. (eds) Bioinformatics. Methods in Molecular Biology, vol 1525. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6622-6_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6622-6_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6620-2

  • Online ISBN: 978-1-4939-6622-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics