Skip to main content

SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing

  • Protocol
  • First Online:
Molecular Methods for Evolutionary Genetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 772))


Next-generation sequencing technologies are revolutionizing the field of evolutionary biology, opening the possibility for genetic analysis at scales not previously possible. Research in population genetics, quantitative trait mapping, comparative genomics, and phylogeography that was unthinkable even a few years ago is now possible. More importantly, these next-generation sequencing studies can be performed in organisms for which few genomic resources presently exist. To speed this revolution in evolutionary genetics, we have developed Restriction site Associated DNA (RAD) genotyping, a method that uses Illumina next-generation sequencing to simultaneously discover and score tens to hundreds of thousands of single-nucleotide polymorphism (SNP) markers in hundreds of individuals for minimal investment of resources. In this chapter, we describe the core RAD-seq protocol, which can be modified to suit a diversity of evolutionary genetic questions. In addition, we discuss bioinformatic considerations that arise from unique aspects of next-generation sequencing data as compared to traditional marker-based approaches, and we outline some general analytical approaches for RAD-seq and similar data. Despite considerable progress, the development of analytical tools remains in its infancy, and further work is needed to fully quantify sampling variance and biases in these data types.

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

Access this chapter

USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
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

Similar content being viewed by others


  1. Mardis ER (2008) The impact of next-­generation sequencing technology on genetics. Trends Genet 24:133141

    Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Asmann YW, Wallace MB, Thompson EA (2008) Transcriptome profiling using next-generation sequencing. Gastroenterology 135:1466–1468

    Article  PubMed  CAS  Google Scholar 

  4. Marguerat S, Wilhelm BT, Bahler J (2008) Next-generation sequencing: applications beyond genomes. Biochem Soc Trans 36:1091–1096

    Article  PubMed  CAS  Google Scholar 

  5. Mortazavi A, Williams BA, McCue K et al (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628

    Article  PubMed  CAS  Google Scholar 

  6. Rokas A, Abbot P (2009) Harnessing genomics for evolutionary insights. Trends Ecol Evol 24:192–200

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  8. Van Tassell CP, Smith TP, Matukumalli LK et al (2008) SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nat Methods 5:247–252

    Article  PubMed  Google Scholar 

  9. Baird NA, Etter PD, Atwood TS et al (2008) Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS ONE 3:e3376

    Article  PubMed  Google Scholar 

  10. Hohenlohe P, Bassham S, Stiffler N et al (2010) Population genomics of parallel adaptation in threespine stickleback using sequenced RAD tags. PLoS Genet 6:e1000862

    Article  PubMed  Google Scholar 

  11. Emerson KJ, Merz CR, Catchen JM et al (2010) Resolving post-glacial phylogeography using high throughput sequencing. Proc Natl Acad Sci USA 107:16196200

    Google Scholar 

  12. Gompert Z, Lucas LK, Fordyce JA et al (2010) Secondary contact between Lycaeides idas and L. melissa in the Rocky Mountains: extensive admixture and a patchy hybrid zone. Mol Ecol 19:3171–3192

    Article  PubMed  CAS  Google Scholar 

  13. Coyne KJ, Burkholder JM, Feldman RA et al (2004) Modified serial analysis of gene expression method for construction of gene expression profiles of microbial eukaryotic species. Appl Environ Microbiol 70:5298–5304

    Article  PubMed  CAS  Google Scholar 

  14. Quail MA, Kozarewa I, Smith F et al (2008) A large genome center’s improvements to the Illumina sequencing system. Nat Methods 5:1005–1010

    Article  PubMed  CAS  Google Scholar 

  15. Lynch M (2009) Estimation of allele frequencies from high-coverage genome-sequencing projects. Genetics 18:295–301

    Article  Google Scholar 

Download references


The authors thank the University of Oregon researchers who, over the past 3 years, have helped troubleshoot many preliminary ­versions of this protocol. This work was funded by grants from the National Institutes of Health (1R24GM079486-01A1 and Ruth L. Kirschstein National Research Service Award F32 GM078949) and the National Science Foundation (IOS-0642264 and DEB-0919090).

Author information

Authors and Affiliations


Corresponding author

Correspondence to William A. Cresko .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Etter, P.D., Bassham, S., Hohenlohe, P.A., Johnson, E.A., Cresko, W.A. (2012). SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing. In: Orgogozo, V., Rockman, M. (eds) Molecular Methods for Evolutionary Genetics. Methods in Molecular Biology, vol 772. Humana Press.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-227-4

  • Online ISBN: 978-1-61779-228-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics