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

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

Abstract

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.

Citation

Workman, R. E., Tang, A. D., Tang, P. S., Jain, M., Tyson, J. R., Razaghi, R., Zuzarte, P. C., Gilpatrick, T., Payne, A., Quick, J., Sadowski, N., Holmes, N., de Jesus, J. G., Jones, K. L., Soulette, C. M., Snutch, T. P., Loman, N., Paten, B., Loose, M., Simpson, J. T., …Timp, W. (2019). Nanopore native RNA sequencing of a human poly(A) transcriptome. Nature Methods, https://doi.org/10.1038/s41592-019-0617-2

• Journal Article Type: Article
• Acceptance Date: Sep 19, 2019
• Online Publication Date: Nov 18, 2019
• Publication Date: Nov 18, 2019
• Deposit Date: Nov 19, 2019
• Publicly Available Date: May 19, 2020
• Journal: Nature Methods
• Print ISSN: 1548-7091
• Electronic ISSN: 1548-7105
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• DOI: https://doi.org/10.1038/s41592-019-0617-2
• Keywords: Biotechnology; Cell Biology; Biochemistry; Molecular Biology
• Public URL: https://nottingham-repository.worktribe.com/output/3330879
• Publisher URL: https://www.nature.com/articles/s41592-019-0617-2
• Contract Date: Nov 19, 2019