Abstract
Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, J. M., Utterback, T. R., Nagle, J. W., Fields, C., and Venter, J. C. (1992) Sequence identification of 2,375 human brain genes. Nature 355, 632–4.
Adams, M. D., Kelley, J. M., Gocayne, J. D., Dubnick, M., Polymeropoulos, M. H., Xiao, H., Merril, C. R., Wu, A., Olde, B., Moreno, R. F., and et al. (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1651–6.
Adams, M. D., Kerlavage, A. R., Fleischmann, R. D., Fuldner, R. A., Bult, C. J., Lee, N. H., Kirkness, E. F., Weinstock, K. G., Gocayne, J. D., White, O., and et al. (1995) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 377, 3–174.
Adams, M. D., Soares, M. B., Kerlavage, A. R., Fields, C., and Venter, J. C. (1993) Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library. Nat Genet 4, 373–80.
Berry, R., Stevens, T. J., Walter, N. A., Wilcox, A. S., Rubano, T., Hopkins, J. A., Weber, J., Goold, R., Soares, M. B., and Sikela, J. M. (1995) Gene-based sequence-tagged-sites (STSs) as the basis for a human gene map. Nat Genet 10, 415–23.
Hillier, L. D., Lennon, G., Becker, M., Bonaldo, M. F., Chiapelli, B., Chissoe, S., Dietrich, N., DuBuque, T., Favello, A., Gish, W., Hawkins, M., Hultman, M., Kucaba, T., Lacy, M., Le, M., Le, N., Mardis, E., Moore, B., Morris, M., Parsons, J., Prange, C., Rifkin, L., Rohlfing, T., Schellenberg, K., Marra, M., and et al. (1996) Generation and analysis of 280,000 human expressed sequence tags. Genome Res 6, 807–28.
Houlgatte, R., Mariage-Samson, R., Duprat, S., Tessier, A., Bentolila, S., Lamy, B., and Auffray, C. (1995) The Genexpress Index: a resource for gene discovery and the genic map of the human genome. Genome Res 5, 272–304.
Khan, A. S., Wilcox, A. S., Polymeropoulos, M. H., Hopkins, J. A., Stevens, T. J., Robinson, M., Orpana, A. K., and Sikela, J. M. (1992) Single pass sequencing and physical and genetic mapping of human brain cDNAs. Nat Genet 2, 180–5.
Okubo, K., Hori, N., Matoba, R., Niiyama, T., Fukushima, A., Kojima, Y., and Matsubara, K. (1992) Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nat Genet 2, 173–9.
Soares, M. B., and Bonaldo, M. F. (1998) Construction and screening of normalized cDNA libraries. Genome Analysis II, 49–157.
Bonaldo, M. F., Lennon, G., and Soares, M. B. (1996) Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res 6, 791–806.
Soares, M. B., Bonaldo, M. F., Jelene, P., Su, L., Lawton, L., and Efstratiadis, A. (1994) Construction and characterization of a normalized cDNA library. Proc Natl Acad Sci USA 91, 9228–32.
Scheetz, T. E., Laffin, J. J., Berger, B., Holte, S., Baumes, S. A., Brown, R., 2nd, Chang, S., Coco, J., Conklin, J., Crouch, K., Donohue, M., Doonan, G., Estes, C., Eyestone, M., Fishler, K., Gardiner, J., Guo, L., Johnson, B., Keppel, C., Kreger, R., Lebeck, M., Marcelino, R., Miljkovich, V., Perdue, M., Qui, L., Rehmann, J., Reiter, R. S., Rhoads, B., Schaefer, K., Smith, C., Sunjevaric, I., Trout, K., Wu, N., Birkett, C. L., Bischof, J., Gackle, B., Gavin, A., Grundstad, A. J., Mokrzycki, B., Moressi, C., O'Leary, B., Pedretti, K., Roberts, C., Robinson, N. L., Smith, M., Tack, D., Trivedi, N., Kucaba, T., Freeman, T., Lin, J. J., Bonaldo, M. F., Casavant, T. L., Sheffield, V. C., and Soares, M. B. (2004) High-throughput gene discovery in the rat. Genome Res 14, 733–41.
Hackett, J. D., Scheetz, T. E., Yoon, H. S., Soares, M. B., Bonaldo, M. F., Casavant, T. L., and Bhattacharya, D. (2005) Insights into a dinoflagellate genome through expressed sequence tag analysis. BMC Genomics 6, 80.
Hackett, J. D., Yoon, H. S., Soares, M. B., Bonaldo, M. F., Casavant, T. L., Scheetz, T. E., Nosenko, T., and Bhattacharya, D. (2004) Migration of the plastid genome to the nucleus in a peridinin dinoflagellate. Curr Biol 14, 213–8.
Bishop, J. O., Morton, J. G., Rosbash, M., and Richardson, M. (1974) Three abundance classes in HeLa cell messenger RNA. Nature 250, 199–204.
Davidson, E. H., and Britten, R. J. (1979) Regulation of gene expression: possible role of repetitive sequences. Science 204, 1052–9.
Trivedi, N., Bischof, J., Davis, S., Pedretti, K., Scheetz, T. E., Braun, T. A., Roberts, C. A., Robinson, N. L., Sheffield, V. C., Soares, M. B., and Casavant, T. L. (2002) Parallel creation of non-redundant gene indices from partial mRNA transcripts. Future Gener Comp Syst 18, 863–70.
LaJeunesse, T. C., Lambert, G., Anderson, R. A., Coffroth, M. A., and Galbraith, D.W. (2005) Symbiodinium (Pyrrhophyta) genome sizes (DNA content) are smallest among dinoflagellates. J Phycol 41, 880–86.
Scheetz, T. E., Trivedi, N., Roberts, C. A., Kucaba, T., Berger, B., Robinson, N. L., Birkett, C. L., Gavin, A. J., O'Leary, B., Braun, T. A., Bonaldo, M. F., Robinson, J. P., Sheffield, V. C., Soares, M. B., and Casavant, T. L. (2003) ESTprep: preprocessing cDNA sequence reads. Bioinformatics 19, 1318–24.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Soares, M.B., de Fatima Bonaldo, M., Hackett, J.D., Bhattacharya, D. (2009). Expressed Sequence Tags: Normalization and Subtraction of cDNA Libraries . In: Parkinson, J. (eds) Expressed Sequence Tags (ESTs). Methods in Molecular Biology, vol 533. Humana Press. https://doi.org/10.1007/978-1-60327-136-3_6
Download citation
DOI: https://doi.org/10.1007/978-1-60327-136-3_6
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-759-4
Online ISBN: 978-1-60327-136-3
eBook Packages: Springer Protocols