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.
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
 | 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. |
 | 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. |
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.
Selected Resources
- Beaulaurier, John et al. (2019) Deciphering bacterial epigenomes using modern sequencing technologies. Nature reviews. Genetics
- Hiraoka, Satoshi et al. (2019) Metaepigenomic analysis reveals the unexplored diversity of DNA methylation in an environmental prokaryotic community. Nature communications
- Ameur, Adam et al. (2019) Single-Molecule Sequencing: Towards Clinical Applications. Trends in biotechnology
- Chen, Yujiao et al. (2019) Study of the whole genome, methylome and transcriptome of Cordyceps militaris. Scientific reports
- Beaulaurier, John et al. (2018) Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation. Nature biotechnology
- Kumari, Romika et al. (2018) PausePred and Rfeet: webtools for inferring ribosome pauses and visualizing footprint density from ribosome profiling data. RNA
- Atack, John M et al. (2018) Phasevarions of bacterial pathogens: Methylomics sheds new light on old enemies. Trends in microbiology
- Johnston, Christopher D et al. (2018) SyngenicDNA: stealth-based evasion of restriction-modification barriers during bacterial genetic engineering BioRxiv
- Poulter, Russell T. M. et al. (2018) Comparison between complete genomes of an isolate of Pseudomonas syringae pv. actinidiae from Japan and a New Zealand isolate of the pandemic. Scientific reports
- Zhao, Liang et al. (2018) The highly heterogeneous methylated genomes and diverse restriction-modification systems of bloom-forming Microcystis. Harmful algae
- Frank, Jeroen et al. (2018) Resolving the complete genome of Kuenenia stuttgartiensis from a membrane bioreactor enrichment using Single-Molecule Real-Time sequencing. Scientific reports
- Yadav, Vikas et al. (2018) RNAi is a critical determinant of centromere evolution in closely related fungi. Proceedings of the National Academy of Sciences of the United States of America
- Ardui, Simon et al. (2018) Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic acids research
- Kjærbølling, Inge et al. (2018) Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species. Proceedings of the National Academy of Sciences of the United States of America
- Phelan, Jody et al. (2018) Methylation in Mycobacterium tuberculosis is lineage specific with associated mutations present globally. Scientific reports
- Nell, Sandra et al. (2017) Genome and methylome variation in Helicobacter pylori with a cag pathogenicity island during early stages of human infection. Gastroenterology
- Deptula, Paulina et al. (2017) De novo assembly of genomes from long sequence reads reveals uncharted territories of Propionibacterium freudenreichii. BMC genomics
- Edwards, Joan E et al. (2017) PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: Insights, challenges andopportunities. Frontiers in microbiology
- Forde, Brian M. et al. (2017) SMRT Sequencing reveals differential patterns of methylation in two O111:H- Shiga toxigenic Escherichia coli isolates from a historic hemolytic uremic syndrome outbreak in Australia BioRxiv
- Costa, Stephen K et al. (2017) Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis. Journal of bacteriology
- Zautner, Andreas E et al. (2017) Monitoring microevolution of OXA-48-producing Klebsiella pneumoniae ST147 in a hospital setting by SMRT Sequencing Journal of antimicrobial chemotherapy
- Utturkar, Sagar M et al. (2017) A case study into microbial genome assembly gap sequences and finishing strategies. Frontiers in microbiology
- Nakano, Kazuma et al. (2017) Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area. Human cell
- Mondo, Stephen J et al. (2017) Widespread adenine N6-methylation of active genes in fungi. Nature genetics
- Elghraoui, Afif et al. (2017) SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis. BMC genomics
- Chi, Kelly Rae et al. (2017) The RNA code comes into focus. Nature
- Cooper, Laurie P et al. (2017) DNA target recognition domains in the Type I restriction and modification systems of Staphylococcus aureus. Nucleic acids research
- Chen, Poyin et al. (2017) Comparative genomics reveals the diversity of restriction-modification systems and DNA methylation sites in Listeria monocytogenes. Applied and environmental microbiology
- Blow, Matthew J et al. (2016) The epigenomic landscape of prokaryotes. PLoS genetics
- Roberts, Richard J et al. (2015) REBASE–a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic acids research
- Korlach, Jonas et al. (2012) Going beyond five bases in DNA sequencing. Current opinion in structural biology
- Flusberg, Benjamin A et al. (2010) Direct detection of DNA methylation during single-molecule, real-time sequencing. Nature methods
- Turner, Stephen (2017) Webinar: An introduction to PacBio’s long-read sequencing & how it has been used to make important scientific discoveries
- Auvignen, Petra (2017) AGBT Virtual Poster: Interspecies interation amoung meat spoilage-related lactic acid bacteria
- (2017) Tutorial: Base modification detection, base modification and motif analysis application [SMRT Link v5.0.0]
- Application Brief: Microbial whole genome sequencing – Best Practices (2019)
- Case Study: Sequencing an historic bacterial collection for the future (2019)
- PacBio Certified Service Providers (2019)
- SMRT Sequencing Brochure: Delivering highly accurate long reads to drive discovery in life science (2019)
- SMRT Analysis Brochure: Gain a deeper understanding of your sequencing data (2018)
- Industrial Biotechnology Brochure: Fuel biotech discovery with confident characterization of microbes and their communities (2016)
- Epigenetics Application Brochure: Characterize the epigenetic landscape of your genome (2016)