Skip to main content

An Introduction to High-Throughput Sequencing Experiments: Design and Bioinformatics Analysis

  • Protocol
  • First Online:
Deep Sequencing Data Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1038))

Abstract

The dramatic fall in the cost of DNA sequencing has revolutionized the experiments within reach in the life sciences. Here we provide an introduction for the domains of analyses possible using high-throughput sequencing, distinguishing between “counting” and “reading” applications. We discuss the steps in designing a high-throughput sequencing experiment, introduce the most widely used applications, and describe basic sequencing concepts. We review the various software programs available for many of the bioinformatics analysis required to make sense of the sequencing data. We hope that this introduction will be accessible to biologists with no previous background in bioinformatics, yet with a keen interest in applying the power of high-throughput sequencing in their research.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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

Institutional subscriptions

References

  1. Goh Y, Fullwood MJ, Poh HM, Peh SQ, Ong CT, Zhang J, Ruan X, Ruan Y (2012) Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) for mapping chromatin interactions and understanding transcription regulation. J Vis Exp e3770

    Google Scholar 

  2. Toung JM, Morley M, Li M, Cheung VG (2011) RNA-sequence analysis of human B-cells. Genome Res 21:991–998

    Article  PubMed  CAS  Google Scholar 

  3. Koehler R, Issac H, Cloonan N, Grimmond SM (2011) The uniqueome: a mappability resource for short-tag sequencing. Bioinformatics 27:272–274

    Article  PubMed  CAS  Google Scholar 

  4. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26

    Article  PubMed  CAS  Google Scholar 

  5. Paszkiewicz K, Studholme DJ (2010) De novo assembly of short sequence reads. Brief Bioinform 11:457–472

    Article  PubMed  CAS  Google Scholar 

  6. Schatz MC, Witkowski J, McCombie WR (2012) Current challenges in de novo plant genome sequencing and assembly. Genome Biol 13:243

    PubMed  CAS  Google Scholar 

  7. McIntyre LM, Lopiano KK, Morse AM, Amin V, Oberg AL, Young LJ, Nuzhdin SV (2011) RNA-seq: technical variability and sampling. BMC Genomics 12:293

    Article  PubMed  CAS  Google Scholar 

  8. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515

    Article  PubMed  CAS  Google Scholar 

  9. Kharchenko PV, Tolstorukov MY, Park PJ (2008) Design and analysis of ChIP-seq experiments for DNA-binding proteins. Nat Biotechnol 26:1351–1359

    Article  PubMed  CAS  Google Scholar 

  10. Auerbach RK, Euskirchen G, Rozowsky J, Lamarre-Vincent N, Moqtaderi Z, Lefrancois P, Struhl K, Gerstein M, Snyder M (2009) Mapping accessible chromatin regions using Sono-Seq. Proc Natl Acad Sci USA 106:14926–14931

    Article  PubMed  CAS  Google Scholar 

  11. Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K et al (2010) De novo assembly of human genomes with massively parallel short read sequencing. Genome Res 20:265–272

    Article  PubMed  CAS  Google Scholar 

  12. Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol I (2009) ABySS: a parallel assembler for short read sequence data. Genome Res 19:1117–1123

    Article  PubMed  CAS  Google Scholar 

  13. Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829

    Article  PubMed  CAS  Google Scholar 

  14. Butler J, MacCallum I, Kleber M, Shlyakhter IA, Belmonte MK, Lander ES, Nusbaum C, Jaffe DB (2008) ALLPATHS: de novo assembly of whole-genome shotgun microreads. Genome Res 18:810–820

    Article  PubMed  CAS  Google Scholar 

  15. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760

    Article  PubMed  CAS  Google Scholar 

  16. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

    Article  PubMed  Google Scholar 

  17. Chen K, Wallis JW, McLellan MD, Larson DE, Kalicki JM, Pohl CS, McGrath SD, Wendl MC, Zhang Q, Locke DP et al (2009) BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Nat Methods 6:677–681

    Article  PubMed  CAS  Google Scholar 

  18. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079

    Article  PubMed  Google Scholar 

  19. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M et al (2010) The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303

    Article  PubMed  CAS  Google Scholar 

  20. Li H, Ruan J, Durbin R (2008) Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18:1851–1858

    Article  PubMed  CAS  Google Scholar 

  21. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q et al (2010) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652

    Article  Google Scholar 

  22. Langmead B, Hansen KD, Leek JT (2010) Cloud-scale RNA-sequencing differential expression analysis with Myrna. Genome Biol 11:R83

    Article  PubMed  Google Scholar 

  23. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W et al (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol 9:R137

    Article  PubMed  Google Scholar 

  24. Feng J, Liu T, Zhang Y (2011) Using MACS to identify peaks from ChIP-Seq data. Curr Protoc Bioinformatics. Chapter 2:Unit 2 14

    Google Scholar 

  25. Zang C, Schones DE, Zeng C, Cui K, Zhao K, Peng W (2009) A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 25:1952–1958

    Article  PubMed  CAS  Google Scholar 

  26. Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA, Handsaker RE, Lunter G, Marth GT, Sherry ST et al (2011) The variant call format and VCFtools. Bioinformatics 27:2156–2158

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Normand, R., Yanai, I. (2013). An Introduction to High-Throughput Sequencing Experiments: Design and Bioinformatics Analysis. In: Shomron, N. (eds) Deep Sequencing Data Analysis. Methods in Molecular Biology, vol 1038. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-514-9_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-514-9_1

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-513-2

  • Online ISBN: 978-1-62703-514-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics