Large-Scale Sequencing and Analytical Processing of ESTs
Expressed sequence tags (ESTs) have proven to be one of the most rapid and cost-effective routes to gene discovery for eukaryotic genomes. Furthermore, their multipurpose uses, such as in probe design for microarrays, determining alternative splicing, verifying open reading frames, and confirming exon/intron and gene boundaries, to name a few, further justify their inclusion in many genomic characterization projects. Hence, there has been a constant increase in the number of ESTs deposited into the dbEST division of GenBank. This trend also correlates to ever-improving molecular techniques for obtaining biological material, performing RNA extraction, and constructing cDNA libraries, and predominantly to ever-evolving sequencing chemistry and instrumentation, as well as to decreased sequencing costs. This chapter describes large-scale sequencing of ESTs on two distinct platforms: the ABI 3730xl and the 454 Life Sciences GS20 sequencers, and the corresponding processes of sequence extraction, processing, and submissions to public databases. While the conventional 3730xl sequencing process is described, starting with the plating of an already-existing cDNA library, the section on 454 GS20 pyrosequencing also includes a method for generating full-length cDNA sequences. With appropriate bioinformatics tools, each of these platforms either used independently or coupled together provide a powerful combination for comprehensive exploration of an organism’s transcriptome.
Key wordsExpressed sequence tags EST sequencing cDNA capillary sequencing ABI 3730xl pyrosequencing GS20
The authors would like to thank the Genome Sequencing Center’s Sequence Production and Technology Development Groups. The authors are supported by grants from the National Human Genome Research Institute U54 HG003079 and the National Institute of Allergy and Infectious Disease AI 46593.
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