ABSTRACT
High throughput cDNA sequencing technologies have dramatically 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 because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes from nanopore RNA data. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology.
DISCLOSURES MA holds shares in Oxford Nanopore Technologies (ONT). MA is a paid consultant to ONT. REW, WT, TG, JRT, JQ, NJL, JTS, NS, AB, MA, HEO, MJ, and ML received reimbursement for travel, accommodation and conference fees to speak at events organised by ONT. NL has received an honorarium to speak at an ONT company meeting. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore. JTS, ML and MA received research funding from ONT.
ACKNOWLEDGEMENTS
The authors are grateful for support from the following individuals. Libby Snell, Botond Sipos and Dan Turner (ONT) provided materials and advice relevant to the 3′ poly(A) standards used to test nanopolish-polya. Daniel Garalde (ONT) provided early advice on use of the MinION for RNA sequencing. Nicholas Conrad gave insight into the correlation of intron retention and poly(A) tail length. Mark Diekhans reviewed the isoform analysis. The authors thank Andrew Beggs, Louise Tee and Tom Nieto (University of Birmingham, UK) for providing cell cultures used in the Birmingham sequencing runs. The project was supported by the following grants: NIH HG010053 (AB, BP, & MA), NIH 5T32HG008345 (AT), NIH HG009190 (WT, JTS), NIH U54HG007990 (BP), U01 HL137183-02 (BP), Oxford Nanopore Research Grant SC20130149 (MA), National Institutes of Health Research Surgical Reconstruction and Microbiology Research Centre (JQ), Medical Research Council CLIMB Fellowship (NL), Wellcome Trust 204843/Z/16/Z (ML), BBSRC BB/N017099/1 and BB/M020061/1 (ML), the Canada Research Chair in Biotechnology and Genomics-Neurobiology (TPS), the Canadian Institutes of Health Research (#10677; TPS), the Canadian Epigenetics, Environment and Health Research Consortium (TPS), the Koerner Foundation (TPS), the Ontario Institute for Cancer Research through funds provided by the Government of Ontario (JTS).
