Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes.

Chloé Baum,Thomas C Evans,Brian P Anton,Bo Yan,Yu-Cheng Lin,Alexey Fomenkov,Lixin Chen,Richard J Roberts,Laurence Ettwiller,Andrew C Tolonen

doi:10.1093/nar/gkab705

Abstract

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.

Highlights

DNA modifications catalysed by DNA methyltransferases are considered to be the most abundant form of epigenetic modification in genomes of both prokaryotes and eukaryotes
This distinction between blocking and mutagenic damage forms the basis of the RIMS-seq method, allowing the identification of methylase specificity based on an elevated number of reads containing C to T transitions at methylated sites (Figure 1A)
The deamination levels typically obtained with RIMS-seq does not permit the quantitative measurement of methylation at each genomic site but rather provides a global methylation signal characteristic of the methylase specificity

Summary

Introduction

DNA modifications catalysed by DNA methyltransferases are considered to be the most abundant form of epigenetic modification in genomes of both prokaryotes and eukaryotes. DNA methylation has been mainly described as part of the sequence-specific restriction modification system (RM), a bacterial immune system to resist invasion of foreign DNA [1]. Contrary to eukaryotes where the position of the m5C methylation is variable and subject to epigenetic states, bacterial methylations tend to be constitutively present at specific sites across the genome. These sites are defined by the methylase specificity and, in the case of RM systems, tend to be fully methylated to avoid cuts by the cognate restriction enzyme. The methylase recognition specificities typically vary from four to eight nucleotides and are often, but not always, palindromic [4]

Methods

Results

Conclusion