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Sequencing of first-strand cDNA library reveals full-length transcriptomes.

ABSTRACT: Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the underrepresentation of both the 5' and 3' ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 5' and 3' ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does not involve second-strand cDNA synthesis, as we Directly Ligate sequencing Adaptors to the First-strand cDNA (DLAF). This novel method with fewer enzymatic reactions results in a higher quality of the libraries than the conventional method. Sequencing of DLAF libraries followed by a novel analysis pipeline enables the profiling of both 5' ends and polyadenylation sites at near-base resolution. Therefore, DLAF offers the first genomics tool to obtain the 'full-length' transcriptome with a single library.

SUBMITTER: Agarwal S

PROVIDER: S-EPMC5054741 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Sequencing of first-strand cDNA library reveals full-length transcriptomes.

Agarwal Saurabh S Macfarlan Todd S TS Sartor Maureen A MA Iwase Shigeki S

Nature communications 20150121

Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the underrepresentation of both the 5' and 3' ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 5' and 3' ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does ...[more]

PMID: 25607527

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Project description:BackgroundJatropha curcas L. is promoted as an important non-edible biodiesel crop worldwide. Jatropha oil, which is a triacylglycerol, can be directly blended with petro-diesel or transesterified with methanol and used as biodiesel. Genetic improvement in jatropha is needed to increase the seed yield, oil content, drought and pest resistance, and to modify oil composition so that it becomes a technically and economically preferred source for biodiesel production. However, genetic improvement efforts in jatropha could not take advantage of genetic engineering methods due to lack of cloned genes from this species. To overcome this hurdle, the current gene discovery project was initiated with an objective of isolating as many functional genes as possible from J. curcas by large scale sequencing of expressed sequence tags (ESTs).ResultsA normalized and full-length enriched cDNA library was constructed from developing seeds of J. curcas. The cDNA library contained about 1 × 10(6) clones and average insert size of the clones was 2.1 kb. Totally 12,084 ESTs were sequenced to average high quality read length of 576 bp. Contig analysis revealed 2258 contigs and 4751 singletons. Contig size ranged from 2-23 and there were 7333 ESTs in the contigs. This resulted in 7009 unigenes which were annotated by BLASTX. It showed 3982 unigenes with significant similarity to known genes and 2836 unigenes with significant similarity to genes of unknown, hypothetical and putative proteins. The remaining 191 unigenes which did not show similarity with any genes in the public database may encode for unique genes. Functional classification revealed unigenes related to broad range of cellular, molecular and biological functions. Among the 7009 unigenes, 6233 unigenes were identified to be potential full-length genes.ConclusionsThe high quality normalized cDNA library was constructed from developing seeds of J. curcas for the first time and 7009 unigenes coding for diverse biological functions including oil biosynthesis were identified. These genes will serve as invaluable genetic resource for crop improvement in jatropha to make it an ideal and profitable crop for biodiesel production.

Dataset Information

Sequencing of first-strand cDNA library reveals full-length transcriptomes.

Publications

Sequencing of first-strand cDNA library reveals full-length transcriptomes.

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