Dataset Information

Genome annotation by high-throughput 5' RNA end determination.

ABSTRACT: Complete gene identification and annotation, including alternative transcripts, remains a challenge in understanding genome organization. Such annotation can be achieved by a combination of computational analysis and experimental confirmation. Here, we describe a high-throughput technique, trans-spliced exon coupled RNA end determination (TEC-RED), that identifies 5' ends of expressed genes in nematodes. TEC-RED can distinguish coding regions from regulatory regions and identify genes as well as their alternative transcripts that have different 5' ends. Application of TEC-RED to approximately 10% of the Caenorhabditis elegans genome yielded tags 75% of which experimentally verified predicted 5'-RNA ends and 25% of which provided previously unknown information about 5'-RNA ends, including the identification of 99 previously unknown genes and 32 previously unknown operons. This technique will be applicable in any organisms that have a trans-splicing reaction from spliced leader RNA. We also describe an efficient sequential method for concatenating short sequence tags for any serial analysis of gene expression-like techniques.

SUBMITTER: Hwang BJ

PROVIDER: S-EPMC341809 | biostudies-other | 2004 Feb

REPOSITORIES: biostudies-other

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Genome annotation by high-throughput 5' RNA end determination.

Hwang Byung Joon BJ Müller Hans-Michael HM Sternberg Paul W PW

Proceedings of the National Academy of Sciences of the United States of America 20040202 6

Complete gene identification and annotation, including alternative transcripts, remains a challenge in understanding genome organization. Such annotation can be achieved by a combination of computational analysis and experimental confirmation. Here, we describe a high-throughput technique, trans-spliced exon coupled RNA end determination (TEC-RED), that identifies 5' ends of expressed genes in nematodes. TEC-RED can distinguish coding regions from regulatory regions and identify genes as well as ...[more]

PMID: 14757812

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Project description:BACKGROUND: Growing interest in cellulolytic clostridia with potential for consolidated biofuels production is mitigated by low conversion of raw substrates to desired end products. Strategies to improve conversion are likely to benefit from emerging techniques to define molecular systems biology of these organisms. Clostridium stercorarium DSM8532T is an anaerobic thermophile with demonstrated high ethanol production on cellulose and hemicellulose. Although several lignocellulolytic enzymes in this organism have been well-characterized, details concerning carbohydrate transporters and central metabolism have not been described. Therefore, the goal of this study is to define an improved whole genome sequence (WGS) for this organism using in-depth molecular profiling by RNA-seq transcriptomics and tandem mass spectrometry-based proteomics. RESULTS: A paired-end Roche/454 WGS assembly was closed through application of an in silico algorithm designed to resolve repetitive sequence regions, resulting in a circular replicon with one gap and a region of 2 kilobases with 10 ambiguous bases. RNA-seq transcriptomics resulted in nearly complete coverage of the genome, identifying errors in homopolymer length attributable to 454 sequencing. Peptide sequences resulting from high-throughput tandem mass spectrometry of trypsin-digested protein extracts were mapped to 1,755 annotated proteins (68% of all protein-coding regions). Proteogenomic analysis confirmed the quality of annotation and improvement pipelines, identifying a missing gene and an alternative reading frame. Peptide coverage of genes hypothetically involved in substrate hydrolysis, transport and utilization confirmed multiple pathways for glycolysis, pyruvate conversion and recycling of intermediates. No sequences homologous to transaldolase, a central enzyme in the pentose phosphate pathway, were observed by any method, despite demonstrated growth of this organism on xylose and xylan hemicellulose. CONCLUSIONS: Complementary omics techniques confirm the quality of genome sequence assembly, annotation and error-reporting. Nearly complete genome coverage by RNA-seq likely indicates background DNA in RNA extracts, however these preps resulted in WGS enhancement and transcriptome profiling in a single Illumina run. No detection of transaldolase by any method despite xylose utilization by this organism indicates an alternative pathway for sedoheptulose-7-phosphate degradation. This report combines next-generation omics techniques to elucidate previously undefined features of substrate transport and central metabolism for this organism and its potential for consolidated biofuels production from lignocellulose.

Dataset Information

Genome annotation by high-throughput 5' RNA end determination.

Publications

Genome annotation by high-throughput 5' RNA end determination.

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