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Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing.

ABSTRACT: Aspergillus oryzae, an important filamentous fungus used in food fermentation and the enzyme industry, has been shown through genome sequencing and various other tools to have prominent features in its genomic composition. However, the functional complexity of the A. oryzae transcriptome has not yet been fully elucidated. Here, we applied direct high-throughput paired-end RNA-sequencing (RNA-Seq) to the transcriptome of A. oryzae under four different culture conditions. With the high resolution and sensitivity afforded by RNA-Seq, we were able to identify a substantial number of novel transcripts, new exons, untranslated regions, alternative upstream initiation codons and upstream open reading frames, which provide remarkable insight into the A. oryzae transcriptome. We were also able to assess the alternative mRNA isoforms in A. oryzae and found a large number of genes undergoing alternative splicing. Many genes and pathways that might be involved in higher levels of protein production in solid-state culture than in liquid culture were identified by comparing gene expression levels between different cultures. Our analysis indicated that the transcriptome of A. oryzae is much more complex than previously anticipated, and these results may provide a blueprint for further study of the A. oryzae transcriptome.

SUBMITTER: Wang B

PROVIDER: S-EPMC2926611 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing.

Wang Bin B Guo Guangwu G Wang Chao C Lin Ying Y Wang Xiaoning X Zhao Mouming M Guo Yong Y He Minghui M Zhang Yong Y Pan Li L

Nucleic acids research 20100414 15

Aspergillus oryzae, an important filamentous fungus used in food fermentation and the enzyme industry, has been shown through genome sequencing and various other tools to have prominent features in its genomic composition. However, the functional complexity of the A. oryzae transcriptome has not yet been fully elucidated. Here, we applied direct high-throughput paired-end RNA-sequencing (RNA-Seq) to the transcriptome of A. oryzae under four different culture conditions. With the high resolution ...[more]

PMID: 20392818

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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.

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Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing.

Publications

Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing.

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