title: Accurate identification and analysis of human mRNA isoforms using deep long read sequencing.
abstract: Precise identification of RNA-coding regions and transcriptomes of eukaryotes is a significant problem in biology. Currently, eukaryote transcriptomes are analyzed using deep short-read sequencing experiments of complementary DNAs. The resulting short-reads are then aligned against a genome and annotated junctions to infer biological meaning. Here we use long-read complementary DNA datasets for the analysis of a eukaryotic transcriptome and generate two large datasets in the human K562 and HeLa S3 cell lines. Both data sets comprised at least 4 million reads and had median read lengths greater than 500 bp. We show that annotation-independent alignments of these reads provide partial gene structures that are very much in-line with annotated gene structures, 15% of which have not been obtained in a previous de novo analysis of short reads. For long-noncoding RNAs (i.e., lncRNA) genes, however, we find an increased fraction of novel gene structures among our alignments. Other important aspects of transcriptome analysis, such as the description of cell type-specific splicing, can be performed in an accurate, reliable and completely annotation-free manner, making it ideal for the analysis of transcriptomes of newly sequenced genomes. Furthermore, we demonstrate that long read sequence can be assembled into full-length transcripts with considerable success. Our method is applicable to all long read sequencing technologies.
authors: Department of Genetics, Stanford University, Stanford, California 94305, USA.
authors: Hagen
authors: H
authors: Tilgner
authors: Debasish
authors: D
authors: Raha
authors: Lukas
authors: L
authors: Habegger
authors: Mohammed
authors: M
authors: Mohiuddin
authors: Mark
authors: M
authors: Gerstein
authors: Michael
authors: M
authors: Snyder
mesh_terms: D017398:Alternative Splicing; D002874:Chromosome Mapping; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005091:Exons; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D020014:K562 Cells; D058977:Molecular Sequence Annotation; D059368:RNA Isoforms / Q000032:analysis* / Q000235:genetics; D062085:RNA, Long Noncoding / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D012270:Ribosomes / Q000235:genetics; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA; D059467:Transcriptome
chemical_list: D018076:DNA, Complementary; D059368:RNA Isoforms; D062085:RNA, Long Noncoding
keywords: RNA; Roche sequencing; human; splicing; transcriptome