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Massively parallel sequencing of the polyadenylated transcriptome of C. elegans.

ABSTRACT: Using massively parallel sequencing by synthesis methods, we have surveyed the polyA+ transcripts from four stages of the nematode Caenorhabditis elegans to an unprecedented depth. Using novel statistical approaches, we evaluated the coverage of annotated features of the genome and of candidate processed transcripts, including splice junctions, trans-spliced leader sequences, and polyadenylation tracts. The data provide experimental support for >85% of the annotated protein-coding transcripts in WormBase (WS170) and confirm additional details of processing. For example, the total number of confirmed splice junctions was raised from 70,911 to over 98,000. The data also suggest thousands of modifications to WormBase annotations and identify new spliced junctions and genes not part of any WormBase annotation, including at least 80 putative genes not found in any of three predicted gene sets. The quantitative nature of the data also suggests that mRNA levels may be measured by this approach with unparalleled precision. Although most sequences align with protein-coding genes, a small fraction falls in introns and intergenic regions. One notable region on the X chromosome encodes a noncoding transcript of >10 kb localized to somatic nuclei.

SUBMITTER: Hillier LW

PROVIDER: S-EPMC2665784 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Massively parallel sequencing of the polyadenylated transcriptome of C. elegans.

Hillier Ladeana W LW Reinke Valerie V Green Philip P Hirst Martin M Marra Marco A MA Waterston Robert H RH

Genome research 20090130 4

Using massively parallel sequencing by synthesis methods, we have surveyed the polyA+ transcripts from four stages of the nematode Caenorhabditis elegans to an unprecedented depth. Using novel statistical approaches, we evaluated the coverage of annotated features of the genome and of candidate processed transcripts, including splice junctions, trans-spliced leader sequences, and polyadenylation tracts. The data provide experimental support for >85% of the annotated protein-coding transcripts in ...[more]

PMID: 19181841

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Project description:BackgroundThe blood-feeding hookworm Necator americanus infects hundreds of millions of people worldwide. In order to elucidate fundamental molecular biological aspects of this hookworm, the transcriptome of the adult stage of Necator americanus was explored using next-generation sequencing and bioinformatic analyses.Methodology/principal findingsA total of 19,997 contigs were assembled from the sequence data; 6,771 of these contigs had known orthologues in the free-living nematode Caenorhabditis elegans, and most of them encoded proteins with WD40 repeats (10.6%), proteinase inhibitors (7.8%) or calcium-binding EF-hand proteins (6.7%). Bioinformatic analyses inferred that the C. elegans homologues are involved mainly in biological pathways linked to ribosome biogenesis (70%), oxidative phosphorylation (63%) and/or proteases (60%); most of these molecules were predicted to be involved in more than one biological pathway. Comparative analyses of the transcriptomes of N. americanus and the canine hookworm, Ancylostoma caninum, revealed qualitative and quantitative differences. For instance, proteinase inhibitors were inferred to be highly represented in the former species, whereas SCP/Tpx-1/Ag5/PR-1/Sc7 proteins ( = SCP/TAPS or Ancylostoma-secreted proteins) were predominant in the latter. In N. americanus, essential molecules were predicted using a combination of orthology mapping and functional data available for C. elegans. Further analyses allowed the prioritization of 18 predicted drug targets which did not have homologues in the human host. These candidate targets were inferred to be linked to mitochondrial (e.g., processing proteins) or amino acid metabolism (e.g., asparagine t-RNA synthetase).ConclusionsThis study has provided detailed insights into the transcriptome of the adult stage of N. americanus and examines similarities and differences between this species and A. caninum. Future efforts should focus on comparative transcriptomic and proteomic investigations of the other predominant human hookworm, A. duodenale, for both fundamental and applied purposes, including the prevalidation of anti-hookworm drug targets.

Project description:Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples.We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens.We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively).Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.

Dataset Information

Massively parallel sequencing of the polyadenylated transcriptome of C. elegans.

Publications

Massively parallel sequencing of the polyadenylated transcriptome of C. elegans.

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