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RNA. 2020 Jan;26(1):19-28. doi: 10.1261/rna.072785.119. Epub 2019 Oct 17.

Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base-specific resolution.

Lorenz DA#1,2,3, Sathe S#1,2,3, Einstein JM1,2, Yeo GW1,2,3.

Author information

1
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA.
2
Stem Cell Program, University of California San Diego, La Jolla, California 92093, USA.
3
Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, USA.
#
Contributed equally

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 N 6-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.

KEYWORDS:

RNA modifications; m6A; nanopore

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