Characterization of the Human ESC Transcriptome by Hybrid Sequencing

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2013 Nov 28

PMID 24282307

Citations 174

Authors

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

Affiliations

Soon will be listed here.

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.

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