BMC Genomics 2007, 8:363 http://www.biomedcentral.com/1471-2164/8/363 the 1100 nucleotides of sequence data lying within 20 nt of the 3' end of the random primer, i.e. under the non-ran- dom 5' portion of DRP1. These sequences were 49% G+C, indicating that the additional GC rich sequence in the 5' portion of the first strand cDNA primer did not seriously bias the priming process. 74% of the sequenced clones in the self-subtracted libraries contained an A at the sixth nucleotide following the AscI restriction site in the primer, indicating that cDNA synthesis mostly initiated, as designed, at an A (Fig. 1A). Since these data provided an initial indication that the library construction technique was working as expected, we randomly picked and sequenced another 200 colonies from the Danio rerio library. All the Danio rerio and Dro- sophila sequences were then compared to the NCBI non- redundant nucleotide sequence database (Gen- Bank+EMBL+DDBJ+PDB sequences – but no EST, STS, GSS, environmental samples or phase 0, 1 or 2 HTGS sequences) using TBLASTX and to the Ensembl unmasked zebrafish genome (Zv6, Ensemble genebuild, Aug. 2006) using BLASTN set to oligo sensitivity. Of the 257 sequences, 10 sequences showed inconclusive or no matches. The remainder matched genomic DNA to better than 97%, indicating that the genomic source contexts of almost all cDNAs were identified (see Additional file 1). These were then categorized with respect to known or hypothetical transcription units (Fig. 2). 245 of the 247 matches are unique in sequenced population, coming from different loci. Although the source material for these libraries was partially degraded total RNA, 70% of the sequences matched known or hypothetical mRNAs. 17% of these were novel, being either first matches to hypo- thetical mRNA sequence or unknown splice variants of previously identified mRNA transcription loci. 16% of sequences matched hnRNA sequence likely removed dur- ing mRNA splicing – which does not necessarily mean that they are non-functional. Eight matches to known or hypothetical ncRNA loci were identified. These included one match to a large rRNA transcription unit and 5 matches to known or hypothetical ncRNA transcription units encoding miRNAs. A total of 17 matches were out- side either known or predicted transcription units. One of these exhibited discontinuous matches to very closely linked same-strand sequence in the genome, indicative of splicing, and suggested a novel unpredicted transcription unit. A total of 10 clones, some in known or predicted introns and others outside of predicted transcription units exhibited significant (e < 10-9) matches to a large number of loci (>50) dispersed throughout the genome. These may represent undescribed interspersed repeat elements (LINES and SINES) in the zebrafish. FCihgaurraecte2rization of equalized cDNA libraries Characterization of equalized cDNA libraries. 257 cDNA sequences from the Danio rerio and Drosophila mela- nogaster self-subtracted cDNA libraries were used as Gen- Bank and Ensembl database query sequences. Diagnostic properties of near identity (>97%) matches were collected and scored. The labeled columns represent the following library characteristics: mRNA – the fraction of clones matching known or hypothetical mRNA sequences. mRNA (novel) – the fraction of clones representing the first reported matches to hypothetical mRNA sequence or previ- ously unknown splice variants of known mRNA transcription units. hnRNA – the fraction of clones matching sequence that is likely removed during mRNA processing, eg. intron. ncRNA – the fraction of clones matching known or hypo- thetical non-coding RNA transcription units. interspersed repetitive – the fraction of clones matching more than 50 dispersed loci in the genome with probabilities of e < 10-9. gDNA – the fraction of clones matching genomic DNA out- side of any known or predicted transcription unit. no match – the fraction of clones that did not match any known cDNA and could not be assigned an origin in the genome. sense – the fraction of matches in the sense orientation of known or hypothetical transcription units. antisense – the fraction of matches in the antisense orientation of known or hypotheti- cal transcription units. 80% were in the sense orientation. Of the antisense matches, only four could clearly be considered artifacts of cloning. 13 antisense clones were structurally normal, exhibiting the appropriate transitions from cDNA to vec- tor sequences, but were spliced according to their sense strand match. Although this suggests that the antisense orientation is an artifact of cloning, such precisely match- ing non-canonical splicing has been reported for at least one genuine antisense transcript [22]. In 227 of the matches, orientation of the cDNAs could be assigned relative to known or hypothetical loci. Of these, We turn finally to the question of the extent of subtrac- tion. The library data clearly show that the cDNA popula- Page 4 of 7 (page number not for citation purposes)