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.