title: 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.
authors.affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Agarwal
authors.affiliation: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Todd S
authors.initials: TS
authors.lastname: Macfarlan
authors.affiliation: Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Maureen A
authors.initials: MA
authors.lastname: Sartor
authors.affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Iwase
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000196:embryology; D019295:Computational Biology; D004256:DNA Polymerase I / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008297:Male; D051379:Mice; D009841:Oligonucleotides / Q000235:genetics; D026723:Polyadenylation; D012313:RNA / Q000032:analysis / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D017423:Sequence Analysis, RNA; D059467:Transcriptome / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D009841:Oligonucleotides; D012313:RNA; D004256:DNA Polymerase I