Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base-specific resolution
- Daniel A. Lorenz1,2,3,4,
- Shashank Sathe1,2,3,4,
- Jaclyn M. Einstein1,2 and
- Gene W. Yeo1,2,3
- 1Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA
- 2Stem Cell Program, University of California San Diego, La Jolla, California 92093, USA
- 3Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, USA
- Corresponding author: geneyeo{at}ucsd.edu
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↵4 These authors contributed equally to this work.
Abstract
Direct RNA sequencing holds great promise for the de novo identification of RNA modifications at single-coordinate resolution; however, interpretation of raw sequencing output to discover modified bases remains a challenge. Using Oxford Nanopore's direct RNA sequencing technology, we developed a random forest classifier trained using experimentally detected N6-methyladenosine (m6A) sites within DRACH motifs. Our software MINES (m6A Identification using Nanopore Sequencing) assigned m6A methylation status to more than 13,000 previously unannotated DRACH sites in endogenous HEK293T transcripts and identified more than 40,000 sites with isoform-level resolution in a human mammary epithelial cell line. These sites displayed sensitivity to the m6A writer, METTL3, and eraser, ALKBH5, respectively. MINES (https://github.com/YeoLab/MINES.git) enables m6A annotation at single coordinate–level resolution from direct RNA nanopore sequencing.
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Footnotes
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.072785.119.
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Freely available online through the RNA Open Access option.
- Received August 11, 2019.
- Accepted October 11, 2019.
This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
