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PMID: 11544204 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library.

Genome research ·Vol. 11 ·No. 9 ·2001-09-00 ·Pages 1594-602

Clark MD, Hennig S, Herwig R, Clifton SW, Marra MA, Lehrach H, Johnson SL, Group tW, WU-GSCnEST Group

Abstract

The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3' and 14,408 5') from over 16,000 of these clones. Clustering of 10,654 high-quality 3' ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.

MeSH Terms
Animals Expressed Sequence Tags Gene Expression Profiling/methods Gene Library Molecular Sequence Data Nucleic Acid Hybridization/methods Nucleotide Mapping/methods Oligonucleotide Probes/genetics Reference Values Sequence Analysis, DNA/methods Zebrafish/genetics
Chemicals
Oligonucleotide Probes
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Clark M D
Max-Planck-Institut für Molekulare Genetik, 14195 Berlin, Germany. clark@molgen.mpg.de
Hennig S
Herwig R
Clifton S W
Marra M A
Lehrach H
Johnson S L
Group tW
WU-GSCnEST Group
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Article Info
Journal
Genome research
Abbr.
Genome Res
ISSN
1088-9051
DOI
10.1101/gr.186901
Email
clark@molgen.mpg.de
Published
2001-09-00
Pages
1594-602
Language
English
Region
United States
NLM ID
9518021
PMCID
PMC311136
Subset
IM
Grants
NIDDK NIH HHS · R01DK55379 · United States
Databases
GENBANK
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PubMed source DOI: 10.1101/gr.186901 PMC Full Text View journal details
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