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Authors: Au, Kin Fai and Sebastiano, Vittorio and Afshar, Pegah Tootoonchi and Durruthy, Jens Durruthy and Lee, Lawrence and Williams, Brian A and van Bakel, Harm and Schadt, Eric E and Reijo-Pera, Renee A and Underwood, Jason G and Wong, Wing Hung

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.

Journal: Proceedings of the National Academy of Sciences of the United States of America
DOI: 10.1073/pnas.1320101110
Year: 2013

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