Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base-specific resolution
Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base-specific resolution
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DOI：
10.1261/rna.072785.119
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发表时间：
2020-01-01
期刊：
影响因子：
4.5
通讯作者：
Yeo, Gene W.
中科院分区：
文献类型：
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作者：
Lorenz, Daniel A.;Sathe, Shashank;Yeo, Gene W.
直接RNA测序在单坐标分辨率下重新鉴定RNA修饰方面有很大的希望;然而，解释原始测序输出以发现修饰的碱基仍然是一个挑战。使用Oxford Nanopore的直接RNA测序技术，我们开发了一种随机森林分类器，该分类器使用DRACH基序内实验检测到的N-6-甲基腺苷（m（6）A）位点进行训练。我们的软件MINES（使用纳米孔测序的m（6）A鉴定）将m（6）A甲基化状态分配给内源性HEK 293 T转录物中超过13，000个先前未注释的DRACH位点，并在人乳腺上皮细胞系中鉴定了超过40，000个具有同种型水平分辨率的位点。这些位点分别对m（6）A写入器（即胃L3）和擦除器（即ALKBH 5）敏感。MINES（https：//github.com/YeoLab/MINES.git）使得m（6）A注释能够以来自直接RNA纳米孔测序的单个坐标水平分辨率进行。
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 (m(6)A) sites within DRACH motifs. Our software MINES (m(6)A Identification using Nanopore Sequencing) assigned m(6)A 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 m(6)A writer, METTL3, and eraser, ALKBH5, respectively. MINES (https://github.com/YeoLab/MINES.git) enables m(6)A annotation at single coordinate-level resolution from direct RNA nanopore sequencing.