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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cerebral Cortex / embryology
  • Computational Biology
  • DNA Polymerase I / metabolism
  • DNA, Complementary / metabolism
  • Gene Library*
  • Genomics
  • High-Throughput Nucleotide Sequencing / methods*
  • Male
  • Mice
  • Oligonucleotides / genetics
  • Polyadenylation
  • RNA / analysis
  • RNA / metabolism
  • Sequence Analysis, DNA / methods*
  • Sequence Analysis, RNA
  • Transcriptome / genetics*

Substances

  • DNA, Complementary
  • Oligonucleotides
  • RNA
  • DNA Polymerase I

Associated data

  • GEO/GSE63424