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See beyond A, T, G, and C

DNA methylation is an important component of microbial defense against foreign DNA, but Restriction-Modification (R-M) systems can also drive bacterial evolution by increasing double strand breaks and C-T mutations and alter the transcription of genes affecting pathogenicity and virulence.

Directly detect epigenetic modifications with SMRT Sequencing

Single Molecule, Real-Time (SMRT) Sequencing detects DNA modifications by measuring variation in the polymerase kinetics of DNA base incorporation during sequencing, eliminating the need for arduous chemical conversion protocols. With SMRT Sequencing, you have the ability to:

  • Detect genome-wide m6A and m4C R-M system motifs at coverage levels recommended for assembly
  • Determine m6A and m4C methylation status at all genomics positions with marginally higher coverage
  • Obtain complete genomes with annotations for epigenetic modification
  • Reveal phase variation of R-M genes that regulate batteries of genes involved in pathogenesis, host adaption, and antibiotic resistance

Workflow: from DNA to microbial methylome

 

Sample & Library Preparation
Use SMRTbell Template Prep 1.0 and Barcoding Adapter Kits 8A/B to pool microbial genomes, targeting 25-fold or 70-fold coverage per strand for m6A or m4C detection, respectively.

Learn More


Sequencing
Generate whole genome and epigenomic data on multiple microbes in a single 10-hour run on the Sequel System.

Data Analysis
Assemble microbial genomes and annotate R-M system motifs in SMRT Analysis or PacBio DevNet.

Learn More

Spotlight: Microbial DNA methylation signatures detected with SMRT Sequencing improves metagenomic binning and plasmid tracking

Scientists used bacterial DNA methylation profiles as endogenous epigenetics barcodes to computationally bin individual reads and assembled contigs by species or strain. This research also enabled linking of plasmids and other mobile genetic elements to their host genomes to improve the strain-level resolution of metagenomes. Explore this research further:

Beaulaurier, J. et al., (2017). Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation. Nature Biotechnology, ePub ahead of print.

Spotlight: Revealing methylation fingerprints to evade the host defenses that thwart genome engineering

SyngenicDNA is a novel stealth-by-engineering approach: editing out recognition motifs specific to a host’s R-M systems makes foreign DNA invisible. The stealth-by-engineering approach, facilitated by SMRT Sequencing, improves bacterial transformation efficiency by up to 70,000-fold and can unlock myriad applications of bacterial genetic engineering in basic research, industrial biology, synthetic biology, and translational science. Explore this research further:

Johnston, C. D., et al. (2018). SyngenicDNA: stealth-based evasion of restriction-modification barriers during bacterial genetic engineering. BioRxiv, Preprint.

Spotlight: Methylomes - a new frontier in prokaryotic biology

SMRT Sequencing sheds light on the methylomes of 230 prokaryotic organisms, uncovering potentially broader roles of DNA methylation in genome biology. Explore this research further:

Blow, M.J. et al., (2016). The epigenomic landscape of prokaryotes. PLoS Genetics, 12(2), p.e1005854.

To learn more about how you can use SMRT Sequencing for microbial epigenetics, contact us.

Selected Resources