Abstract
Transcriptome sequencing from cDNA libraries has been extensively and efficiently used to analyze sequence variation in protein-coding genes (Expressed Sequence Tags) in eukaryote species. Rapid advances in next-generation sequencing (NGS) technology, in terms of cost, speed, and throughput, allow us to address previously unanswerable questions in the fields of ecology, evolution, and systematics using these genomic tools. Transcriptome sequencing from individuals across different populations and species enables researchers to study the evolution of gene sequence variation at a population genomics level. In this chapter, we describe a customized protocol that has been successfully optimized for the development of normalized cDNA libraries in eukaryote systems suitable for Roche 454 GS FLX sequencing, requiring only small quantities of starting material.
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Acknowledgments
The authors thank James Ford, Jade Buchanan-Carter, Zach Smith, and Keithanne Mockaitis for technical support for sequencing, and Jie Huang for assistance with manuscript preparation. This work was supported in part by a Roche Applied Science/454 Life Sciences 1 GB sequencing grant program to Z.L. and L.H.R., a Natural Sciences and Engineering Research Council of Canada (NSERC) grant #353026 to L.H.R, and the Indiana METACyt Initiative of Indiana University, funded in part through a major grant from the Lilly Endowment, Inc.
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Lai, Z., Zou, Y., Kane, N.C., Choi, JH., Wang, X., Rieseberg, L.H. (2012). Preparation of Normalized cDNA Libraries for 454 Titanium Transcriptome Sequencing. In: Pompanon, F., Bonin, A. (eds) Data Production and Analysis in Population Genomics. Methods in Molecular Biology, vol 888. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-870-2_8
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DOI: https://doi.org/10.1007/978-1-61779-870-2_8
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-869-6
Online ISBN: 978-1-61779-870-2
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