Sequencing Technologies for the Hotspot of Microbial Epigenomics – DNA N6-methyladenine

The most prevalent modified base in bacterial DNA is N6-methyladenine and involves functions in regulating modification (RM) processes, repairing new strand DNA, and controlling gene expression. Virulence and relationships between bacteria and host cells may be caused by methyltransferase mutations and overexpression. No enzymes are capable of demethylating DNA m6A in vivo, but competitive protein binding at target sites can influence methylation and thus modify transcription in newly replicated cells. Frame-shift mutations could also lead to progress in the gene expression involved in cell wall formation and restore pathways at repeat regions in methyltransferase genes. These mechanisms of step variation can help bacteria avoid mammalian host defenses, but it is still unclear if frame-shifts of methyltransferase lead to tolerance to other conditions.

Previous to the advent of single-molecule sequencing, restriction digests or sequencing after immunoprecipitation is used in techniques to assess m6A in DNA. A test is used to differentiate DNA polymerase kinetics involving methylated bases and templates for unmethylated bases, calculated as an inter-pulse period (IPD) in the single-molecule, real-time (SMRT) analysis pipeline for PacBio sequencing results. Various groups have also shown that minor improvements to electrical impulses as DNA passes across nanopores will expose the existence of base modifications using sequencers produced by Oxford Nanopore Technologies (ONT).

Techniques for Sequencing N6-Methyladenine

SMRT sequencing: The SMRT sequencing of PacBio depends on sequencing-by-synthesis, in which the sequence of a circular DNA template is defined from the series of fluorescence pulses, each arising from a polymerase fixed to the bottom of a well that adds one named nucleotide. Therefore, base changes do not change the sequence labeled base, but they change the polymerase's kinetics. Base changes may be derived by the contrast of a changed prototype with an in silico model or an unmodified prototype by assessing the inter-pulse length. For instance, the presence of 6mA in the template strand appears to hinder the polymerase's integration of the complementary T. Kinetic disruption patterns may be more complicated and context-dependent, while base adjustment often depends on the severity of the disturbances. These features make SMRT most sensitively detect 4mC and 6mA, making it useful to bacterial epigenomics. SMRT sequencing has significantly increased the number of identified methyltransferases since 2012. SMRT sequencing has also been used in Leishmania to diagnose base J (β-d-glucosyl-hydroxymethyluracil) and has the ability to analyze unexplained improvements.

Nanopore sequencing: When a single-stranded nucleic acid is tightened along, ONT nanopore sequencing tests the difference of ionic current through a biological nanopore. In a method called base-calling, neural networks convert the present track into nucleotides. Base DNA modifications add raw signal anomalies, make them observable. Nanopolish is a signal-level analysis software kit for Oxford Nanopore sequencing data that can identify 5mCG with a pre-trained algorithm. Since Nanopolish combines a 5mCG model, in addition to the targeted sample, there is no need to sequence a PCR-amplified, unmodified unit. At a single-read, almost single-nucleotide resolution, Nanopolish generates the likelihood that a base is changed.

Three measures are involved in identifying base modifications on nanopore sequencing: (1) base-calling with canonical bases, (2) attaching the raw signal to a genetic reference, and (3) assessing the proof that a base is changed. So far, nanopore sequencing performs better in 5mCG detection, while m6A precision is comparable to PacBio. And also compared to SMRT, the reduced cost per Gb makes it ideal for larger genomes.


  1. O'Brown, Z.K., Boulias, K., Wang, J. et al. Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA. BMC Genomics. 2019, 20, 445.
  2. Quentin Gouil, Andrew Keniry; Latest techniques to study DNA methylation. Essays Biochem. 2019, 63 (6): 639–648.
  3. Chuan-Le Xiao, et al. Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 on the mitochondrial genome, 2018.
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