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 5' expressed sequence tags (5' 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 5' 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.

Figure 1

Graphical display of expressed sequence tag (EST) clusters. (A) is an example of six different subclusters aligning to the Curated Gene CG8977 and numbered SC2-SC7. The subclusters are color coded with respect to the number of EST members as shown. (B) is a gene model based on the merged subclusters illustrating two possible splice variants and numbered SC2-as and SC4-as. SC2-as is a merge of SC2, -3, -5, and -7 and SC4-as is a merge of SC4 and SC6.

Figure 2

CG regions. The observed percentage of each alignment type for clusters representing DGCr2 is indicated in the right column. CG regions are represented as open boxes. CGs are shown numbered in gray boxes (exons) connected by lines (introns). The genomic sequence is shown as a hatched line. Aligned EST clusters are shown in black, and the ESTs chosen for DGC2 are gray with a black border. All CGs and alignments are shown representing one strand of the genome proceeding 5′ to 3′ as indicated in (A). See text for a full description of CG regions.