High-throughput sequencing for 1-methyladenosine (m(1)A) mapping in RNA

Abstract

Detection and mapping of modified nucleotides in RNAs is a difficult and laborious task. Several physico-chemical approaches based on differential properties of modified nucleotides can be used, however, most of these methods do not allow high-throughput analysis. Here we describe in details a method for mapping of rather common 1-methyladenosine (m(1)A) residues using high-throughput next generation sequencing (NGS). Since m(1)A residues block primer extension during reverse transcription (RT), the accumulation of abortive products as well as the nucleotide misincorporation can be detected in the sequencing data. The described library preparation protocol allows to capture both types of cDNA products essential for further bioinformatic analysis. We demonstrate that m(1)A residues produce characteristic arrest and mismatch rates and combination of both can be used for their detection as well as for discrimination of m(1)A from other modified A residues present in RNAs.

Keywords: High-throughput sequencing; Misincorporation signature; RNA modification; Reverse transcription.