Expand the horizon
DNA methylation significantly affects many biological processes in microbes and may play a role in pathogenicity and virulence. Characterizing DNA methylation and methyltransferase specificities is now possible during long-read sequencing.
Single Molecule, Real-Time (SMRT) Sequencing offers direct detection of DNA modifications by measuring variation in the polymerase kinetics of DNA base incorporation during sequencing. The PacBio Systems have the sensitivity to detect epigenetic modification without amplification or chemical conversions. These enhanced capabilities support hypothesis-free analysis of sequences for epigenetic signatures, previously unknown DNA modifications, and methyltransferase recognition motifs throughout bacterial genomes. With SMRT Sequencing, you have the ability to:
- Simultaneously detect multiple types of epigenetic modifications
- Visualize genome-wide methylation with motif analysis at single-base and DNA-strand resolution
- Obtain complete genomes with annotations for epigenetic modification
- Resolve and phase strand-specific modifications
Workflow: from DNA to microbial methylome
- Long-insert library preparation
- Prepare long-insert SMRTbell library (10 kb library prep protocol)
- Size-selection options enrich for longest inserts
- Library input as low as 100 ng of gDNA
- Library automation supported
- SMRT Sequencing with PacBio Systems
- Achieve average read lengths of ~10 kb
- Complete microbial genomes in a single SMRT Cell on the PacBio RS II
- Data analysis with SMRT Analysis or PacBio DevNet
- Use SMRT Analysis epigenomic tools for modification detection and motif analysis
- Find more advanced epigenetic analysis bioinformatic resources on PacBio DevNet
- Watch a 10-minute tutorial on how to assemble complete microbial genomes and characterize methylomes with SMRT Analysis
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: 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
- 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
- Pollard, Martin O et al. (2018) Long reads: their purpose and place. Human Molecular Genetics
- Zhao, Liang et al. (2018) The highly heterogeneous methylated genomes and diverse restriction-modification systems of bloom-forming Microcystis. Harmful Algae
- Slager, Jelle et al. (2018) Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39 BioRxiv
- 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
- Atack, John M et al. (2018) Phasevarions of bacterial pathogens: Methylomics sheds new light on old enemies. Trends in Microbiology
- 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
- Beaulaurier, John et al. (2017) Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation. Nature Biotechnology
- 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
- 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
- Costa, Stephen K et al. (2017) Bypassing the restriction system to improve transformation ofStaphylococcus epidermidis. Journal of Bacteriology
- 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
- Phelan, Jody et al. (2018) Methylation in Mycobacterium tuberculosis is lineage specific with associated mutations present globally. Scientific Reports
- Simões, Patrícia Martins et al. (2016) Single-molecule sequencing (PacBio) of the Staphylococcus capitis NRCS-A clone reveals the basis of multidrug resistance and adaptation to the Neonatal Intensive Care Unit environment. Frontiers in Microbiology
- Seong, Hoon Je et al. (2016) Methylome analysis of two Xanthomonas spp. using Single-Molecule Real-Time Sequencing. The Plant Pathology Journal
- Weigele, Peter et al. (2016) Biosynthesis and function of modified bases in bacteria and their viruses. Chemical Reviews
- Suzuki, Yuta et al. (2016) AgIn: Measuring the landscape of CpG methylation of individual repetitive elements. Bioinformatics
- Dong, Jiaqiang et al. (2016) Analysis of tandem gene copies in maize chromosomal regions reconstructed from long sequence reads. Proceedings of the National Academy of Sciences of the United States of America
- Anjum, Awais et al. (2016) Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168. Nucleic Acids Research
- Davis-Richardson, Austin G et al. (2016) Integrating DNA methylation and gene expression data in the development of the soybean-Bradyrhizobium N2-fixing symbiosis. Frontiers in Microbiology
- Cohen, Nadia R et al. (2016) A role for the bacterial GATC methylome in antibiotic stress survival. Nature Genetics
- Blow, Matthew J et al. (2016) The epigenomic landscape of prokaryotes. PLoS Genetics
- Zhu, Lingxiang et al. (2016) Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology. Nucleic Acids Research
- Roberts, Richard J et al. (2015) REBASE–a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Research
- Poster: Hall, Richard J et al. (2015) A workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data
- Presentation: Morishita, Shinichi et al. (2015) Understanding methylome, metagenome, structural variants using SMRT Sequencing
- Poster: Lambert, Christine et al. (2016) Multiplexing strategies for microbial whole genome SMRT Sequencing
- 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]
- Suzuki, Yoshihiko (2016) AGBT Virtual Poster (Japanese): Meta-methylome of human gut microbiome using SMRT Sequencing
- Blow, Matthew (2015) AGBT Conference: Functional genomics – gene annotation and methylome analysis in bacteria
- Application Brief: Microbial whole genome sequencing – Best Practices. (2018)
- SMRT Sequencing Brochure: Revolutionize genomics with SMRT Sequencing. (2018)
- PacBio Certified Service Providers (2018)
- SMRT Analysis Brochure: Gain a deeper understanding of your sequencing data (2017)
- Case Study: Scientists advance understanding of microbial genomes, epigenomes and communities with SMRT Sequencing. (2016)
- 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)
- Microbiology and Infectious Disease Brochure: Novel insights into microbial and viral complexity. (2015)