Base-resolution detection ofN⁴-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing

Abstract

Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N6-methyladenine (6mA), 5-methylcytosine (5mC) and N4-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

“…In bacteria, directed DNA methylation on N6 of adenine (6mA) regulates mismatch repair, DNA replication and transcription. 4mC has been identified in thermophilic bacteria and archaea, and to a lesser extent in mesophilic bacteria [5][6][7][8][9]. Relatively little is known about the function of 4mC beyond its role in the restriction-modification system, which 6mA also regulates.…”

MASQC: Next Generation Sequencing Assists Third Generation Sequencing for Quality Control in N6-Methyladenine DNA Identification

“…Different types of DNA modification (DNA damage, m5C, and derivatives produced during demethylation) at or adjacent to the sites of interest may produce an IPD ratio similar to that of the adenine site, resulting in a high FPR of 6mA events (Flusberg et al, 2010). In bacterial genomes, DNA methylation is relatively limited in form (6mA, 5mC, 4mC) (Yu et al, 2015) and highly motif driven, which greatly reduces the difficulty of detecting and distinguishing 6mA events from other DNA modifications. In contrast, the 6mA events in the eukaryotic genome are much more abundant and motif is driven weakly, that is why it may coexist with other forms of DNA modifications.…”

A Deep Neural Network for Identifying DNA N4-Methylcytosine Sites

“…However, SMRT sequencing is costly and is not conducive to the analysis of various species. Recently, Yu et al (2015) proposed a method for the determination of methylcytosine in genomic DNA by 4 mC-Tet-assisted bisulfite sequencing, which can accurately generate a genome-wide, single-base resolution map of 4mC, and finally identify the 4mC motif associated with the bacterial R-M system. Biological experiments are laborious and expensive when performing genome-wide testing.…”