PT  - JOURNAL ARTICLE
AU  - Ying Chen
AU  - Nadia M. Davidson
AU  - Yuk Kei Wan
AU  - Harshil Patel
AU  - Fei Yao
AU  - Hwee Meng Low
AU  - Christopher Hendra
AU  - Laura Watten
AU  - Andre Sim
AU  - Chelsea Sawyer
AU  - Viktoriia Iakovleva
AU  - Puay Leng Lee
AU  - Lixia Xin
AU  - Hui En Vanessa Ng
AU  - Jia Min Loo
AU  - Xuewen Ong
AU  - Hui Qi Amanda Ng
AU  - Jiaxu Wang
AU  - Wei Qian Casslynn Koh
AU  - Suk Yeah Polly Poon
AU  - Dominik Stanojevic
AU  - Hoang-Dai Tran
AU  - Kok Hao Edwin Lim
AU  - Shen Yon Toh
AU  - Philip Andrew Ewels
AU  - Huck-Hui Ng
AU  - N.Gopalakrishna Iyer
AU  - Alexandre Thiery
AU  - Wee Joo Chng
AU  - Leilei Chen
AU  - Ramanuj DasGupta
AU  - Mile Sikic
AU  - Yun-Shen Chan
AU  - Boon Ooi Patrick Tan
AU  - Yue Wan
AU  - Wai Leong Tam
AU  - Qiang Yu
AU  - Chiea Chuan Khor
AU  - Torsten Wüstefeld
AU  - Ploy N. Pratanwanich
AU  - Michael I. Love
AU  - Wee Siong Sho Goh
AU  - Sarah B. Ng
AU  - Alicia Oshlack
AU  - Jonathan Göke
AU  - SG-NEx consortium
TI  - A systematic benchmark of Nanopore long read RNA sequencing for transcript level analysis in human cell lines
AID  - 10.1101/2021.04.21.440736
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.04.21.440736
4099  - http://biorxiv.org/content/early/2021/04/22/2021.04.21.440736.short
4100  - http://biorxiv.org/content/early/2021/04/22/2021.04.21.440736.full
AB  - The human genome contains more than 200,000 gene isoforms. However, different isoforms can be highly similar, and with an average length of 1.5kb remain difficult to study with short read sequencing. To systematically evaluate the ability to study the transcriptome at a resolution of individual isoforms we profiled 5 human cell lines with short read cDNA sequencing and Nanopore long read direct RNA, amplification-free direct cDNA, PCR-cDNA sequencing. The long read protocols showed a high level of consistency, with amplification-free RNA and cDNA sequencing being most similar. While short and long reads generated comparable gene expression estimates, they differed substantially for individual isoforms. We find that increased read length improves read-to-transcript assignment, identifies interactions between alternative promoters and splicing, enables the discovery of novel transcripts from repetitive regions, facilitates the quantification of full-length fusion isoforms and enables the simultaneous profiling of m6A RNA modifications when RNA is sequenced directly. Our study demonstrates the advantage of long read RNA sequencing and provides a comprehensive resource that will enable the development and benchmarking of computational methods for profiling complex transcriptional events at isoform-level resolution.Competing Interest StatementThe authors have declared no competing interest.