Finishing a whole-genome shotgun: Release 3 of the Drosophila melanogaster euchromatic genome sequence

BACKGROUND: The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions. RESULTS: Our finishing process was designed to close gaps, improve sequence quality and validate the assembly. Sequence traces derived from the WGS and draft sequencing of individual bacterial artificial chromosomes (BACs) were assembled into BAC-sized segments. These segments were brought to high quality, and then joined to constitute the sequence of each chromosome arm. Overall assembly was verified by comparison to a physical map of fingerprinted BAC clones. In the current version of the 116.9 Mb euchromatic genome, called Release 3, the six euchromatic chromosome arms are represented by 13 scaffolds with a total of 37 sequence gaps. We compared Release 3 to Release 2; in autosomal regions of unique sequence, the error rate of Release 2 was one in 20,000 bp. CONCLUSIONS: The WGS strategy can efficiently produce a high-quality sequence of a metazoan genome while generating the reagents required for sequence finishing. However, the initial method of repeat assembly was flawed. The sequence we report here, Release 3, is a reliable resource for molecular genetic experimentation and computational analysis

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

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. [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.

Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160

The development of a vaccine to provide protective immunity to human immunodeficiency virus type 1 (HIV-1), the virus causing AIDS, would be the most practical method to control its spread. Subunit vaccines consisting of virus envelope glycoproteins, produced by recombinant DNA technology, are effective in preventing viral infections. We have now used this approach in the development of a candidate AIDS vaccine. Chimpanzees were immunized with recombinant forms of the HIV-1 glycoproteins gp120 and gp160 produced in Chinese hamster ovary cells, and then challenged with HIV-1. The control and the two animals immunized with the gp160 variant became infected within 7 weeks of challenge. The two animals immunized with the gp120 variant have shown no signs of infection after more than 6 months. These studies demonstrate that recombinant gp120, formulated in an adjuvant approved for human use, can elicit protective immunity against a homologous strain of HIV-1