Large-scale sequencing and analytical processing of ESTs

Makedonka Mitreva, Elaine R. Mardis

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Scopus citations

Abstract

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.

Original languageEnglish
Title of host publicationExpressed Sequence Tags (ESTs)
Subtitle of host publicationGeneration and Analysis
EditorsJohn Parkinson
Pages153-187
Number of pages35
DOIs
StatePublished - Dec 1 2009

Publication series

NameMethods in Molecular Biology
Volume533
ISSN (Print)1064-3745

Keywords

  • ABI 3730xl
  • EST
  • Expressed sequence tags
  • GS20
  • capillary sequencing
  • pyrosequencing
  • sequencing cDNA

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    Mitreva, M., & Mardis, E. R. (2009). Large-scale sequencing and analytical processing of ESTs. In J. Parkinson (Ed.), Expressed Sequence Tags (ESTs): Generation and Analysis (pp. 153-187). (Methods in Molecular Biology; Vol. 533). https://doi.org/10.1007/978-1-60327-136-3_8