The Drosophila Gene Collection: Identification of Putative Full-Length cDNAs for 70% of D. melanogaster Genes

Abstract

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5Ј expressed sequence tags (5Ј expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ∼40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5Ј ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. BF485518-BF503517, BF503521-BF506780, BG631888-BG631996, BG633696-BG637540, BG640063-BG641469, BI141709-BI142246, BI161485-BI173971, BI212109-BI216987, BI227448-BI233322, BI234009-BI243989, BI351612-BI354228, BI354231-BI355901, BI355935-BI358751, BI361285-BI376197, BI481532-BI487261, BI563331-BI593695, BI604243-BI620155, BI620158-BI635012, BI635064-BI638027, and BI638030-BI642053. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: J. Pringle and M. Fuller.]The identification of all expressed genes and the structure(s) of their transcripts are prerequisites for many structural and functional genomic studies. Gene-finding programs are valuable tools for identifying gene structure, but they are errorprone and suffer from the inability to predict untranslated regions (UTRs) (Ashburner 2000;Reese et al. 2000). Direct analysis of gene transcripts is the only proven way to establish gene structures with confidence. Generating a collection of expressed sequence tags (ESTs) from high quality cDNA libraries is a widely used approach for acquiring this information (Adams et al. 1991). The sequences of ESTs and full-length nonredundant cDNA collections provide ideal tools for genome annotation and for the further training of gene prediction algorithms. Our first D.melanogaster EST project yielded putative full-length clones corresponding to >5000 different genes (Rubin et al. 2000). This was acco...

Genome-wide analysis of promoter architecture in Drosophila melanogaster

“…In an initial assessment of the distributions of TSSs within active promoters in the D. melanogaster embryo, we analyzed 66,169 previously described embryonic cap-trapped 59 ESTs (Stapleton et al 2002), known as RIKEN embryo (RE) ESTs, including 3035 clones represented by full-insert cDNA sequences, to the reference genome sequence (Release 5, http://www.fruitfly.org). Because these ESTs are long sequences (average length 453 nt), >92% (61,429) map uniquely to the genome (Supplemental Table 1).…”

“…In the 0-24-h embryo total RNA sample, we targeted all FB5.12 transcript models that overlap 59 ESTs from the RE (Stapleton et al 2002) and LD (Rubin et al 2000) cDNA libraries, both constructed from mixed-stage embryos. We added transcripts of genes expressed in the embryo based on whole-mount RNA in situ hybridization (Tomancak et al 2007) and literature surveys.…”

“…On the adult total RNA sample, we used 1920 of the primer pairs from the embryo RACE experiments and identical protocols to target FB5.12 transcript models known to be expressed in the adult based on overlap with RH (RIKEN Head) ESTs (Stapleton et al 2002).…”

“…Previously described cap-trapped 59 RE ESTs (Stapleton et al 2002) were reanalyzed to ensure accurate vector trimming and genomic alignment. The vector sequence at the junction in EST reads was identified using cross_match (http://www.phrap.org) and aligned to the genome using sim4 (Florea et al 1998) centered on a region surrounding the BLAST HSP (Altschul et al 1997).…”

“…More recently, cap analysis of gene expression (CAGE), a high-throughput method for promoter discovery, has been used in mouse and human to characterize capped transcript ends (Shiraki et al 2003;Carninci et al 2006). In Drosophila, TSSs have been defined on a modest scale by sequencing and annotation of 59 ESTs generated from cap-trapped cDNA clones (Misra et al 2002;Stapleton et al 2002). The current reference annotation of the D. melanogaster genome sequence uses these data, and 59 ESTs from non-cap-trapped cDNA libraries (Rubin et al 2000), to define 59 transcript ends (Drysdale 2008).…”