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Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing.

ABSTRACT: Defining the transcriptome, the repertoire of transcribed regions encoded in the genome, is a challenging experimental task. Current approaches, relying on sequencing of ESTs or cDNA libraries, are expensive and labor-intensive. Here, we present a general approach for ab initio discovery of the complete transcriptome of the budding yeast, based only on the unannotated genome sequence and millions of short reads from a single massively parallel sequencing run. Using novel algorithms, we automatically construct a highly accurate transcript catalog. Our approach automatically and fully defines 86% of the genes expressed under the given conditions, and discovers 160 previously undescribed transcription units of 250 bp or longer. It correctly demarcates the 5' and 3' UTR boundaries of 86 and 77% of expressed genes, respectively. The method further identifies 83% of known splice junctions in expressed genes, and discovers 25 previously uncharacterized introns, including 2 cases of condition-dependent intron retention. Our framework is applicable to poorly understood organisms, and can lead to greater understanding of the transcribed elements in an explored genome.

SUBMITTER: Yassour M

PROVIDER: S-EPMC2638735 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing.

Yassour Moran M Kaplan Tommy T Fraser Hunter B HB Levin Joshua Z JZ Pfiffner Jenna J Adiconis Xian X Schroth Gary G Luo Shujun S Khrebtukova Irina I Gnirke Andreas A Nusbaum Chad C Thompson Dawn-Anne DA Friedman Nir N Regev Aviv A

Proceedings of the National Academy of Sciences of the United States of America 20090210 9

Defining the transcriptome, the repertoire of transcribed regions encoded in the genome, is a challenging experimental task. Current approaches, relying on sequencing of ESTs or cDNA libraries, are expensive and labor-intensive. Here, we present a general approach for ab initio discovery of the complete transcriptome of the budding yeast, based only on the unannotated genome sequence and millions of short reads from a single massively parallel sequencing run. Using novel algorithms, we automatic ...[more]

PMID: 19208812

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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:BackgroundAnalyses 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.MethodsWe 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.ResultsWe 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).ConclusionWhole-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.

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Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing.

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Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing.

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