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SNP Discovery by Transcriptome Pyrosequencing

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cDNA Libraries

Part of the book series: Methods in Molecular Biology ((MIMB,volume 729))

Abstract

Single nucleotide polymorphisms (SNPs) are single base differences between haplotypes. SNPs are abundant in many species and valuable as markers for genetic map construction, modern molecular breeding programs, and quantitative genetic studies. SNPs are readily mined from genomic DNA or cDNA sequence obtained from individuals having two or more distinct genotypes. While automated Sanger sequencing has become less expensive over time, it is still costly to acquire deep Sanger sequence from several genotypes. “Next-generation” DNA sequencing technologies that utilize new chemistries and massively parallel approaches have enabled DNA sequences to be acquired at extremely high depths of coverage faster and for less cost than traditional sequencing. One such method is represented by the Roche/454 Life Sciences GS-FLX Titanium Series, which currently uses pyrosequencing to produce up to 400–600 million bases of DNA sequence/run (>1 million reads, ∼400 bp/read). This chapter discusses the use of high-throughput pyrosequencing for SNP discovery by focusing on 454 sequencing of maize cDNA, the development of a computational pipeline for polymorphism detection, and the subsequent identification of over 7,000 putative SNPs between Mo17 and B73 maize. In addition, alternative alignment and polymorphism detection strategies that implement Illumina short reads, data processing and visualization tools, and reduced representation techniques that reduce the sequencing of repeat DNA, thus enabling efficient analysis of genome sequence, are discussed.

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Acknowledgments

We thank Sanzhen Liu (Iowa State University), Yi Jia (Iowa State University), and Cheng-Ting Eddy Yeh (Iowa State University) for comments on the manuscript; Drs. Haiyan Wu (Iowa State University and China Agriculture University), Ananth Kalyanaraman (Washington State University), Wei Wu (Iowa State University), and An-Ping Hsia (Iowa State University) for sharing Brachypodium 454 EST data prior to publication; Drs. Richard Buggs (University of Florida), Doug Soltis (University of Florida), and Pam Soltis (University of Florida) for sharing Tragopogon data prior to publication; Dr. Nathan Springer (University of Minnesota) and Dr. Jeff Jeddeloh (Roche NimbleGen Inc.) for sharing maize sequence capture data prior to publication; Dr. Scott Emrich (University of Notre Dame) for stimulating discussions; and Marianne Smith (Iowa State University) and Lisa Coffey (Iowa State University) for technical assistance. This project was supported by competitive grants from the National Science Foundation Plant Genome Program to P.S.S. (DBI-0321711, DBI-0321595, and DBI-0919254) and W.B.B. (DBI-0501758 and DBI-0919254), by the National Research Initiative (NRI) Plant Genome Program of the USDA Cooperative State Research, Education and Extension Service (CSREES) to W.B.B., and by Hatch Act and State of Iowa funds to P.S.S.

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  1. Department of Biology and the Genetics Institute, University of Florida, Gainesville, FL, USA

    W. Brad Barbazuk

  2. Department of Agronomy, Center for Plant Genomics, Iowa State University, Ames, IA, USA

    Patrick S. Schnable

Authors
  1. W. Brad Barbazuk
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  2. Patrick S. Schnable
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Correspondence to W. Brad Barbazuk .

Editor information

Editors and Affiliations

  1. , Dept. Plant Sciences & Plant Pathology, Montana State University, Bozeman, 59717-3150, Montana, USA

    Chaofu Lu

  2. Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, Washington, USA

    John Browse

  3. Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, Washington, USA

    James G. Wallis

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© 2011 Springer Science+Business Media, LLC

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Barbazuk, W.B., Schnable, P.S. (2011). SNP Discovery by Transcriptome Pyrosequencing. In: Lu, C., Browse, J., Wallis, J. (eds) cDNA Libraries. Methods in Molecular Biology, vol 729. Humana Press. https://doi.org/10.1007/978-1-61779-065-2_15

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  • DOI: https://doi.org/10.1007/978-1-61779-065-2_15

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-064-5

  • Online ISBN: 978-1-61779-065-2

  • eBook Packages: Springer Protocols

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