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Nat Commun. 2015 Jan 21;6:6002. doi: 10.1038/ncomms7002.

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

Author information

1
Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA.
2
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
3
Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA.

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.

PMID:
25607527
PMCID:
PMC5054741
DOI:
10.1038/ncomms7002
[Indexed for MEDLINE]
Free PMC Article

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