title: Characterization of the human ESC transcriptome by hybrid sequencing.
abstract: Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, full-length mRNA isoforms are not captured. On the other hand, third-generation sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete.
authors.affiliation: Department of Statistics and Department of Health Research and Policy, Stanford University, Stanford, CA 94305.
authors.forename: Kin Fai
authors.initials: KF
authors.lastname: Au
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sebastiano
authors.forename: Pegah Tootoonchi
authors.initials: PT
authors.lastname: Afshar
authors.forename: Jens Durruthy
authors.initials: JD
authors.lastname: Durruthy
authors.forename: Lawrence
authors.initials: L
authors.lastname: Lee
authors.forename: Brian A
authors.initials: BA
authors.lastname: Williams
authors.forename: Harm
authors.initials: H
authors.lastname: van Bakel
authors.forename: Eric E
authors.initials: EE
authors.lastname: Schadt
authors.forename: Renee A
authors.initials: RA
authors.lastname: Reijo-Pera
authors.forename: Jason G
authors.initials: JG
authors.lastname: Underwood
authors.forename: Wing Hung
authors.initials: WH
authors.lastname: Wong
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000737:chemistry / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D020033:Protein Isoforms / Q000235:genetics*; D059467:Transcriptome / Q000235:genetics*
chemical_list: D020033:Protein Isoforms
keywords: PacBio; alternative splicing; hESC transcriptome; isoform discovery; lncNRA