Preparation of normalized cDNA libraries for 454 Titanium transcriptome sequencing.
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.
The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN, USA. zlai@cgb.indiana.edu
Zhao
Z
Lai
Yi
Y
Zou
Nolan C
NC
Kane
Jeong-Hyeon
JH
Choi
Xinguo
X
Wang
Loren H
LH
Rieseberg
D020224:Expressed Sequence Tags; D015723:Gene Library; D018745:Genome, Plant; D056914:Genomic Structural Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D010944:Plants / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D059467:Transcriptome