Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 110, Issue 50, Pages E4821-E4830Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1320101110
Keywords
isoform discovery; PacBio; hESC transcriptome; alternative splicing; lncNRA
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Funding
- National Human Genome Research Institute [R01HG005717]
- California Institute of Technology Beckman Center for Functional Genomics
- Encode project (National Human Genome Research Institute) [U54 HG004576, U54 HG006998]
- National Heart, Lung, and Blood Institute [U01HL100397]
- National Institute of Child Health and Human Development [U54 HD068158]
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Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, fulllength 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.
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