AceView: a comprehensive cDNA-supported gene and transcripts annotation

BACKGROUND: Regions covering one percent of the genome, selected by ENCODE for extensive analysis, were annotated by the HAVANA/Gencode group with high quality transcripts, thus defining a benchmark. The ENCODE Genome Annotation Assessment Project (EGASP) competition aimed at reproducing Gencode and finding new genes. The organizers evaluated the protein predictions in depth. We present a complementary analysis of the mRNAs, including alternative transcript variants. RESULTS: We evaluate 25 gene tracks from the University of California Santa Cruz (UCSC) genome browser. We either distinguish or collapse the alternative splice variants, and compare the genomic coordinates of exons, introns and nucleotides. Whole mRNA models, seen as chains of introns, are sorted to find the best matching pairs, and compared so that each mRNA is used only once. At the mRNA level, AceView is by far the closest to Gencode: the vast majority of transcripts of the two methods, including alternative variants, are identical. At the protein level, however, due to a lack of experimental data, our predictions differ: Gencode annotates proteins in only 41% of the mRNAs whereas AceView does so in virtually all. We describe the driving principles of AceView, and how, by performing hand-supervised automatic annotation, we solve the combinatorial splicing problem and summarize all of GenBank, dbEST and RefSeq into a genome-wide non-redundant but comprehensive cDNA-supported transcriptome. AceView accuracy is now validated by Gencode. CONCLUSION: Relative to a consensus mRNA catalog constructed from all evidence-based annotations, Gencode and AceView have 81% and 84% sensitivity, and 74% and 73% specificity, respectively. This close agreement validates a richer view of the human transcriptome, with three to five times more transcripts than in UCSC Known Genes (sensitivity 28%), RefSeq (sensitivity 21%) or Ensembl (sensitivity 19%)

Transcriptome sequencing of the Microarray Quality Control (MAQC) RNA reference samples using next generation sequencing

<p>Abstract</p> <p>Background</p> <p>Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC) reference RNA samples using Roche's 454 Genome Sequencer FLX.</p> <p>Results</p> <p>We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values ≤ 10^-20. We measured gene expression levels in the A and B samples by counting the numbers of reads that mapped to individual RefSeq genes in multiple sequencing runs to evaluate the MAQC quality metrics for reproducibility, sensitivity, specificity, and accuracy and compared the results with DNA microarrays and Quantitative RT-PCR (QRTPCR) from the MAQC studies. In addition, 88% of the reads were successfully aligned directly to the human genome using the AceView alignment programs with an average 90% sequence similarity to identify 137,899 unique exon junctions, including 22,193 new exon junctions not yet contained in the RefSeq database.</p> <p>Conclusion</p> <p>Using the MAQC metrics for evaluating the performance of gene expression platforms, the ExpressSeq results for gene expression levels showed excellent reproducibility, sensitivity, and specificity that improved systematically with increasing shotgun sequencing depth, and quantitative accuracy that was comparable to DNA microarrays and QRTPCR. In addition, a careful mapping of the reads to the genome using the AceView alignment programs shed new light on the complexity of the human transcriptome including the discovery of thousands of new splice variants.</p

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants

Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

publication en ligne. Article dans revue scientifique avec comité de lecture. nationale.National audienceThe human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

The Caenorhabditis elegans unc-32 Gene Encodes Alternative Forms of a Vacuolar ATPase a Subunit

International audienceEukaryotes possess multiple isoforms of the a subunit of the V(0) complex of vacuolar-type H(+)-ATPases (V-ATPases). Mutations in the V-ATPase a3 isoform have recently been shown to result in osteopetrosis, a fatal disease in humans, but no function has yet been ascribed to other isoforms. In Caenorhabditis elegans, the unc-32 mutant was originally isolated on the basis of its movement defect. We have isolated four new mutant alleles, the strongest of which is embryonic lethal. We show here that unc-32 corresponds to one of the four genes encoding a V-ATPase a subunit in the nematode, and we present their expression patterns and a molecular analysis of the gene family. unc-32 gives rise via alternative splicing to at least six transcripts. In the uncoordinated alleles, the transcript unc-32 B is affected, suggesting that it encodes an isoform that is targeted to synaptic vesicles of cholinergic neurons, where it would control neurotransmitter uptake or release. Other isoforms expressed widely during embryogenesis are mutated in the lethal alleles and would be involved in other acidic organelles. Our results indicate that V-ATPase a subunit genes are highly regulated and have tissue-specific function