Strategies for undertaking expressed sequence tag (EST) projects

Methods Mol Biol. 2009;533:13-32. doi: 10.1007/978-1-60327-136-3_2.


Complementary DNA (cDNA) sequencing can be used to sample an organism's transcriptome, and the generated EST sequences can be used for a variety of purposes. They are especially important for enhancing the utility of a genome sequence or for providing a gene catalog for a genome that has not or will not be sequenced. In planning and executing a cDNA project, several criteria must be considered. One should clearly define the project purpose, including organism tissue(s) choice, whether those tissues should be pooled, ability to acquire adequate amounts of clean and well-preserved tissue, choice of type(s) of library, and construction of a library (or libraries) that is compatible with project goals. In addition, one must possess the skills to construct the library (or libraries), keeping in mind the number of clones that will be necessary to meet the project requirements. If one is inexperienced in cDNA library construction, it might be wise to outsource the library production and/or sequence and analysis to a sequencing center or to a company that specializes in those activities. One should also be aware that new sequencing platforms are being marketed that may offer simpler protocols that can produce cDNA data in a more rapid and economical manner. Of course, the bioinformatics tools will have to be in place to de-convolute and aid in data analysis for these newer technologies. Possible funding sources for these projects include well-justified grant proposals, private funding, and/or collaborators with available funds.

Publication types

  • Research Support, N.I.H., Extramural
  • Review

MeSH terms

  • Algorithms
  • Ancylostoma / metabolism
  • Animals
  • Cloning, Molecular
  • DNA, Complementary / metabolism
  • Evolution, Molecular
  • Expressed Sequence Tags*
  • Gene Library
  • Genetic Techniques*
  • Genome
  • Nucleic Acid Hybridization
  • Oligonucleotide Array Sequence Analysis
  • Polymorphism, Single Nucleotide
  • RNA, Messenger / metabolism
  • Strongyloides / metabolism


  • DNA, Complementary
  • RNA, Messenger