Nanopore native RNA sequencing of a human poly(A) transcriptome

Rachael E. Workman, Alison D. Tang, Paul S. Tang, Miten Jain, John R. Tyson, Roham Razaghi, Philip C. Zuzarte, Timothy Gilpatrick, Alexander Payne, Joshua Quick, Norah Sadowski, Nadine Holmes, Jaqueline Goes de Jesus, Karen L. Jones, Cameron M. Soulette, Terrance P. Snutch, Nicholas Loman, Benedict Paten, Matthew Loose, Jared T. SimpsonHugh E. Olsen, Angela N. Brooks, Mark Akeson, Winston Timp*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

114 Citations (Scopus)


High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes.

Original languageEnglish
Pages (from-to)1297-1305
Number of pages9
JournalNature Methods
Issue number12
Publication statusPublished - 1 Dec 2019
Externally publishedYes

Bibliographical note

Funding Information:
The authors are grateful for support from the following individuals. L. Snell, B. Sipos and D. Turner (ONT) provided materials and advice relevant to the 3′ poly(A) standards used to test nanopolish-polya. D. Garalde (ONT) provided early advice on use of the MinION for RNA sequencing. N. Conrad gave insight into the correlation of intron retention and poly(A) tail length. M. Diekhans reviewed the isoform analysis. Z. M. Chrzanowska-Lightowlers, T. Suzuki and S. Okada commented on early drafts of the manuscript. A. Beggs, L. Tee and T. Nieto (University of Birmingham, UK) provided cell cultures used in the Birmingham sequencing runs. The project was supported by the following grants: NIH HG010053 (A.N.B., B.P. and M.A.), NIH 5T32HG008345 (A.D.T.), NIH HG010538 (W.T.), NIH U54HG007990 (B.P.), U01 HL137183-02 (B.P.), Oxford Nanopore Research Grant SC20130149 (M.A.), National Institutes of Health Research Surgical Reconstruction and Microbiology Research Centre (J.Q.), Medical Research Council CLIMB Fellowship (N.L.), Wellcome Trust 204843/Z/16/Z (M.L.), BBSRC BB/ N017099/1 and BB/M020061/1 (M.L.), the Canada Research Chair in Biotechnology and Genomics-Neurobiology (T.P.S.), the Canadian Institutes of Health Research (no. 10677; T.P.S.), the Canadian Epigenetics, Environment and Health Research Consortium (T.P.S.), the Koerner Foundation (T.P.S.), Genome Canada (OGI-136, J.T.S.), and the Ontario Institute for Cancer Research through funds provided by the Government of Ontario (J.T.S.), Pew Charitable Trust (A.N.B.).

Funding Information:
M.A. holds options in Oxford Nanopore Technologies (ONT). M.A. is a paid consultant to ONT. R.E.W., W.T., T.G., J.R.T., J.Q., N.J.L., J.T.S., N.S., A.N.B., M.A., H.E.O., M.J. and M.L. received reimbursement for travel, accommodation and conference fees to speak at events organised by ONT. N.L. has received an honorarium to speak at an ONT company meeting. W.T. has two patents (8,748,091 and 8,394,584) licensed to ONT. M.A. is an inventor on 11 UC patents licensed to ONT (6,267,872, 6,465,193, 6,746,594, 6,936,433, 7,060,50, 8,500,982, 8,679,747, 9,481,908, 9,797,013, 10,059,988, and 10,081,835). J.T.S., M.L. and M.A. received research funding from ONT.

Publisher Copyright:
© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc.


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