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

Saurabh Agarwal, Todd S. Macfarlan, Maureen A. Sartor and Shigeki Iwase ()
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Saurabh Agarwal: University of Michigan
Todd S. Macfarlan: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health
Maureen A. Sartor: University of Michigan
Shigeki Iwase: University of Michigan

Nature Communications, 2015, vol. 6, issue 1, 1-12

Abstract: 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.

Date: 2015
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DOI: 10.1038/ncomms7002

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