Characterization of the human ESC transcriptome by hybrid sequencing

作者：Kin Fai Au, Vittorio Sebastiano, Pegah Tootoonchi Afshar, Jens Durruthy Durruthy, Lawrence Lee, Brian A. Williams, Harm van Bakel, Eric E. Schadt, Renee A. Reijo Pera, Jason G. Underwood, Wing Hung Wong · 发表于：Proceedings of the National Academy of Sciences · 年份：2013 · DOI：10.1073/pnas.1320101110 · 被引用次数：389 · 研究领域：Cancer-related molecular mechanisms research、RNA Research and Splicing、RNA modifications and cancer

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.