Industrial microbiologists rely on comprehensive genomic information to identify and develop complex biological products. Single Molecule, Real-Time (SMRT) Sequencing delivers a more complete view of individual organisms and microbial communities, fueling research for modern pharmaceutical discovery, environmental remediation, chemical commodity production, and agriculture products.
Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices, but most applications are focused on conserved core genomic regions due to limitations of short-read technologies. In this study we established a long-read technology-based WGS screening program of all first-episode MRSA blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads revealed widespread gain/loss of accessory mobile genetic elements among established hospital- and community-associated lineages impacting >10% of each genome, and frequent megabase-scale inversions between endogenous prophages. We also characterized an outbreak of a CC5/ST105/USA100 clone among 3…
DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (?RSM strain) of a putative Type I restriction modification system lost all…
Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression,…
Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23…
Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C…
Brian Anton from New England BioLabs presents data on methylation analysis using SMRT Sequencing. He describes both restriction-modification systems and orphan methylases, noting that the number of methylases characterized has more than tripled since the introduction of SMRT Sequencing. The presentation includes a phylogenetic analysis of methyltransferase genes
Yoshihiko Suzuki, Graduate Student from University of Tokyo presents his poster (in Japanese) on characterizing a methylome of the human gut microbiome using SMRT Sequencing and metagenomic assembly
Shiga toxin-producing Escherichia coli (STEC) is an emerging pathogen. Recently there has been a global in the number of outbreaks caused by non-O157 STECs, typically involving six serogroups O26, O45, 0103, 0111, and 0145. STEC O145:H28 has been associated with severe human disease including hemolytic-uremic syndrome (HUS), and is demonstrated by the 2007 Belgian ice-cream-associated outbreak and 2010 US lettuce-associated outbreak, with over 10% of patients developing HUS in each. The goal of this work was to do comparative genomics of strains, clinical and environmental, to investigate genome diversity and virulence evolution of this important foodborne pathogen.
The data throughput of next-generation sequencing allows whole microbial communities to be analyzed using a shotgun sequencing approach. Because a key task in taking advantage of these data is the ability to cluster reads that belong to the same member in a community, single-molecule long reads of up to 30 kb from SMRT Sequencing provide a unique capability in identifying those relationships and pave the way towards finished assemblies of community members. Long reads become even more valuable as samples get more complex with lower intra-species variation, a larger number of closely related species, or high intra-species variation. Here we…
The throughput of SMRT Sequencing and long reads allows microbial communities to be analyzed using a shotgun sequencing approach. Key to leveraging this data is the ability to cluster sequences belonging to the same member of a community. Long reads of up to 40 kb provide a unique capability in identifying those relationships, and pave the way towards finished assemblies of community members. Long reads are highly valuable when samples are more complex and containing lower intra-species variation, such as a larger number of closely related species, or high intra-species variation. Here, we present a collection of tools tailored for…
It has recently become possible to rapidly and accurately detect epigenetic signatures in bacterial genomes using third generation sequencing data. Monitoring the speed at which a single polymerase inserts a base in the read strand enables one to infer whether a modification is present at that specific site on the template strand. These sites can be challenging to detect in the absence of high coverage and reliable reference genomes.Here we provide a new method for detecting epigenetic motifs in bacteria on datasets with low-coverage, with incomplete references, and with mixed samples (i.e. metagenomic data). Our approach treats motif inference as…
Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs…
In attempt to obtain a thermophilic host for the conversion of lignocellulose derived substrates into lactic acid, Geobacillus thermodenitrificans T12 was isolated from a compost heap. It was selected from over 500 isolates as a genetically tractable hemicellulolytic lactic acid producer, requiring little nutrients. The strain is able to ferment glucose and xylose simultaneously and can produce lactic acid from xylan, making it a potential host for biotechnological applications. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 and 56 kb. It has a total of 3.676 genes with an average genomic GC content of…
Until recently, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.All KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each…