A computational and experimental approach to validating annotations and gene predictions in theDrosophila melanogastergenome

Abstract

Five years after the completion of the sequence of the Drosophila melanogaster genome, the number of protein-coding genes it contains remains a matter of debate; the number of computational gene predictions greatly exceeds the number of validated gene annotations. We have assembled a collection of >10,000 gene predictions that do not overlap existing gene annotations and have developed a process for their validation that allows us to efficiently prioritize and experimentally validate predictions from various sources by sequencing RT-PCR products to confirm gene structures. Our data provide experimental evidence for 122 proteincoding genes. Our analyses suggest that the entire collection of predictions contains only Ϸ700 additional protein-coding genes. Although we cannot rule out the discovery of genes with unusual features that make them refractory to existing methods, our results suggest that the D. melanogaster genome contains Ϸ14,000 protein-coding genes.gene number ͉ validation ͉ genome annotation T he total number of protein-coding genes in the Drosophila melanogaster genome remains a subject of debate. Whereas those who curated the D. melanogaster genome concluded that the annotated 13,659 genes in the 3.1 release likely constitute 95% of all protein-coding genes (1), others researchers have concluded that many, possibly thousands, of protein-coding genes remain unannotated (2). Two issues have fueled the debate surrounding gene number in D. melanogaster: the large numbers of computational gene predictions located within intergenic regions and varying standards of experimental evidence for concluding that a gene prediction corresponds to a real gene.As of release 3.1, Ϸ50% of the D. melanogaster genome is intergenic. Running the gene prediction program GENSCAN (3) on every intergenic region in the D. melanogaster genome results in 10,644 gene predictions spread amongst 62 megabases (Mb) of annotation-free sequence. Surely some of these predictions are real, but how many? The best way to answer this question is to subject a representative sample of the gene predictions to some validation procedure.The design and interpretation of experiments intended to assay expression of genes that have been predicted computationally have become controversial. One approach is to rely on hybridization to microarrays or RT-PCR assays for transcript expression (2), with the detection of a product by agarose gel electrophoresis taken as confirmation of the corresponding gene prediction. However, as our results show, unless the diagnostic PCR product includes a splice junction, amplification of residual genomic DNA and detection of unprocessed transcripts may lead to false verifications of gene predictions. It has also been critical to determine the sequence, and not just the size, of the PCR products (4).One way to obtain spliced cDNAs for sequencing is to perform RT-PCR with a 3Ј-oligo(dT) primer and an upstream PCR primer located in the prediction's 5Ј-most exon. The advantages of this approach are that it requires only a ...

Selective constraint on noncoding regions of hominid genomes

“…To address this possibility, we used transcript predictions from ab initio prediction programs [17,18] in order to eliminate all upstream sequences that contained predicted transcripts (see Materials and Methods). These programs have a relatively liberal definition of genes [19], which allowed us to be more stringent in identifying sequences that do not contain genes in them. Our more stringent set included 2,390 genes.…”

MAKER: An easy-to-use annotation pipeline designed for emerging model organism genomes

“…Gene number is one such comparison. Though protein-coding gene numbers have been a subject of controversy, most annotated model Eukaryotes contain on the order of 15,000-25,000 protein-coding genes (for discussion, see Yandell et al 2005). Drosophila, e.g., is believed to contain fewer than 15,000 protein coding genes , and the WS160 WormBase release puts the number of C. elegans genes at slightly less than 20,000.…”

Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures

“…Comparative information has improved computational gene predictors 5 , but their accuracy still falls far short of well-studied gene catalogues such as the FlyBase annotation, which combines computational gene prediction 37 , high-throughput experimental data [38][39][40][41][42] and extensive manual curation 23 . Recognizing this, we set out not only to produce an independent computational annotation of protein-coding genes in the fly genome, but also to assess and refine its already high-quality annotations 43 .…”