An Oligonucleotide Fingerprint Normalized and Expressed Sequence Tag Characterized Zebrafish cDNA Library

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 3Ј and 14,408 5Ј) from over 16,000 of these clones. Clustering of 10,654 high-quality 3Ј 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.

A framework for integrating the songbird brain

“…This approach has not yet been applied to songbirds, resulting in a gap between songbird and mammalian, fish, and insect molecular research (Soares 1997;Churchill and Oliver 2001;Clark et al 2001;Miki et al 2001). To narrow this molecular gap, we have formed a consortium of investigators from Duke University in the USA and RIKEN Institute in Japan, that has the following aims: (1) clone full-length cDNAs from normalized zebra finch brain libraries that represent mRNAs expressed during commonly studied vocal communication states, (2) characterize these cDNAs by sequencing, and (3) use them to generate non-redundant cDNA microarrays for asking biological questions.…”

Mutation ofweak atrium/atrial myosin heavy chaindisrupts atrial function and influences ventricular morphogenesis in zebrafish

“…To identify a zebrafish amhc gene, we evaluated the expression patterns of available zebrafish ESTs (RZPD, Berlin) resembling myosin heavy chain genes, found one that was atrium-specific (fc52a03) (Clark et al, 2001), and cloned a corresponding full-length cDNA using previously described reverse transcription and RACE techniques (Keegan et al, 2002). To detect amhc mutations, we amplified fragments of amhc cDNA from mutant embryos using previously described RT-PCR strategies (Keegan et al, 2002).…”

Gene Discovery in the Apicomplexa as Revealed by EST Sequencing and Assembly of a Comparative Gene Database

“…] The generation of expressed sequence tags (ESTs) provides a rapid means of gene discovery from single-pass sequencing of randomly selected cDNAs. This approach has been particularly useful for complex, model genomes including human (Hillier et al 1996), rat (Scheetz et al 2001), mouse (Marra et al 1999b), fish (Clark et al 2001), and rice (Ewing et al 1999). One of the primary advantages of ESTs is that the identification of putative genes by BLAST comparisons (Altschul et al 1990) enables researchers to begin biological analyses prior to the completion, or even initiation, of a full genome sequence.…”