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Microsatellites pp 89–99Cite as

Next-Generation Sequencing for High-Throughput Molecular Ecology: A Step-by-Step Protocol for Targeted Multilocus Genotyping by Pyrosequencing

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1006))

Abstract

Next-generation sequencing technology can now provide population biologists and phylogeographers with information at the genomic scale; however, many pertinent questions in population genetics and phylogeography can be answered effectively with modest levels of genomic information. For the past two decades, most population-level studies have lacked nuclear DNA (nDNA) sequence data due to the complications and cost of amplifying and sequencing diploid loci. However, pyrosequencing of emulsion PCR reactions, amplifying from only one molecule at a time, can generate megabases of clonally amplified loci at high coverage, thereby greatly simplifying allelic sequence determination. Here, we present a step-by-step methodology for utilizing the 454 GS FLX Titanium pyrosequencing platform to simultaneously sequence 16 populations (at 20 individuals per population) at 10 different nDNA loci (3,200 loci in total) in one plate of sequencing for less than the cost of traditional Sanger sequencing.

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Acknowledgments

The authors would like to thank Maria Byrne, Sergio Barbosa, Carson Keever, Jason Addison, Michael Hart, and Richard Grosberg for their extensive collaborative and supportive efforts with this project. We also thank Clarissa Murch for extensive help with project organization and lab work. Lastly, we would like to thank Scott Hunicke-Smith of University of Texas for his vast assistance with implementing this unique 454 sequencing project and the Hawai’i Institute of Marine Biology EPSCoR core genetics facility. This project was funded a grant from the National Science Foundation (Bio-OCE 0623699). This is contribution #1520 from the Hawai’i Institute of Marine Biology and 8754 from the School of Ocean and Earth Sciences and Technology (SOEST).

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Authors and Affiliations

  1. Hawai‘i Institute of Marine Biology, University of Hawai‘I, Kāne‘ohe, HI, USA

    Jonathan B. Puritz & Robert J. Toonen

  2. Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX, USA

    Jonathan B. Puritz

Authors
  1. Jonathan B. Puritz
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  2. Robert J. Toonen
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Editor information

Editors and Affiliations

  1. , Department of Plant, Soil,, Southern Illinois University, Carbondale, Lincoln Drive 1205, Carbondale, 62901, Illinois, USA

    Stella K. Kantartzi

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Puritz, J.B., Toonen, R.J. (2013). Next-Generation Sequencing for High-Throughput Molecular Ecology: A Step-by-Step Protocol for Targeted Multilocus Genotyping by Pyrosequencing. In: Kantartzi, S. (eds) Microsatellites. Methods in Molecular Biology, vol 1006. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-389-3_6

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  • DOI: https://doi.org/10.1007/978-1-62703-389-3_6

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-388-6

  • Online ISBN: 978-1-62703-389-3

  • eBook Packages: Springer Protocols

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