AUTHOR=Cheng Yin-Wei , Chen Yun-Mei , Zhao Qian-Qian , Zhao Xing , Wu Ya-Ru , Chen Dan-Ze , Liao Lian-Di , Chen Yang , Yang Qian , Xu Li-Yan , Li En-Min , Xu Jian-Zhen 

TITLE=Long Read Single-Molecule Real-Time Sequencing Elucidates Transcriptome-Wide Heterogeneity and Complexity in Esophageal Squamous Cells

JOURNAL=Frontiers in Genetics

VOLUME=Volume 10 - 2019

YEAR=2019

URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2019.00915

DOI=10.3389/fgene.2019.00915

ISSN=1664-8021

ABSTRACT=Esophageal squamous cell carcinoma is a leading cause of cancer death. Mapping the transcriptional landscapes such as isoforms, fusion transcripts as well as long noncoding RNAs, have played a central role to understand the regulating mechanism during malignant processes. However, canonical methods such as short-read RNA-seq are difficult to define the entire polyadenylated RNA molecules. Here we combined single-molecule real-time sequencing with RNA-seq to generate high-quality long reads, and to survey the transcriptional program in esophageal squamous cells. Compared with the recent annotations of human transcriptome (Ensemble 38 release 91), SMRT data identified many unannotated transcripts, novel isoforms of known genes and an expanding repository of long intergenic noncoding RNAs. By integrating with annotation of lincRNA catalog, 1521 esophageal cancer specific lincRNAs were defined from SMRT reads. Isoform usage analysis revealed the shifted alternative splicing patterns, which can be recaptured from clinical samples or supported by previous studies. Utilizing vigorous heuristics criteria, we also detected multiple transcript fusions, which are not documented in current gene fusion database or readily identified from RNA-seq reads. Two novel fusion transcripts were verified based on RT-PCR and Sanger sequencing. Overall, our long-reads single molecule sequencing largely expands current understanding of full length transcriptome in esophageal cells, and provides novel insights on the transcriptional diversity during oncogenic transformation.