With Single Molecule, Real-Time (SMRT) Sequencing and the Sequel Systems, you can affordably assemble reference-quality microbial genomes that are >99.999% (Q50) accurate.
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.
Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single-nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non- pathogenic to pathogenic…
2015 SMRT Informatics Developers Conference Presentation Slides: Shinichi Morishita of the University of Tokyo presented on how his team has been using SMRT Sequencing to better understand methylomes, metagenomes and structural variation of various eukaryotic genomes.
Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus. Our analyses revealed marked differences in the gene arrangement, chromosomal location and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive…
Two extremely halophilic archaea, namely, Natrinema versiforme BOL5-4 and Natrinema pallidum BOL6-1, were isolated from a Bolivian salt mine and their genomes sequenced using single-molecule real-time sequencing. The GC-rich genomes of BOL5-4 and BOL6-1 were 4.6 and 3.8 Mbp, respectively, with large chromosomes and multiple megaplasmids. Genome annotation was incorporated into HaloWeb and methylation patterns incorporated into REBASE.Copyright © 2019 DasSarma et al.
Here, we report the complete genome sequence and full methylome analysis of a newly isolated, aerobic, thermophilic, Gram-positive actinomycete, a strain of Thermoactinomyces vulgaris designated strain 2H.Copyright © 2019 Mankai et al.
The chromosomal methylation statuses of the highly virulent Vibrio vulnificus strain CMCP6 grown in human serum and in seawater are compared here. Growth in seawater resulted in ~4 times as much methylation as that in human serum, primarily N4-methylcytosines.Copyright © 2019 Conrad and Harwood.
The genomes of two extremely halophilic Archaea species, Haloarcula marismortui and Haloferax mediterranei, were sequenced using single-molecule real-time sequencing. The ~4-Mbp genomes are GC rich with multiple large plasmids and two 4-methyl-cytosine patterns. Methyl transferases were incorporated into the Restriction Enzymes Database (REBASE), and gene annotation was incorporated into the Haloarchaeal Genomes Database (HaloWeb).Copyright © 2019 DasSarma et al.
Micrococcus luteus has been found in a wide range of habitats. We report the complete genome sequence and methylome analysis of strain SA211 isolated from a hypersaline, lithium-rich, high-altitude salt flat in Argentina with single-molecule real-time sequencing.
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,…
Ethanol-type fermentation is one of three main fermentation types in the acidogenesis of anaerobic treatment systems. Non-spore-forming Ethanoligenens is as a typical genus capable of ethanol-type fermentation in mixed culture (i.e. acetate-ethanol fermentation). This genus can produce ethanol, acetate, CO2, and H2 using carbohydrates, and has application potential in anaerobic bioprocesses. Here, the complete genome sequences and methylome of Ethanoligenens harbinense strains with different autoaggregative and coaggregative abilities were obtained using the PacBio single-molecule real-time sequencing platform. The genome size of E. harbinense strains was about 2.97-3.10?Mb with 55.5% G+C content. 3020-3153 genes were annotated, most of which were methylated…
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…
The complete genome of Cordyceps militaris was sequenced using single-molecule real-time (SMRT) sequencing technology at a coverage over 300×. The genome size was 32.57?Mb, and 14 contigs ranging from 0.35 to 4.58?Mb with an N50 of 2.86?Mb were assembled, including 4 contigs with telomeric sequences on both ends and an additional 8 contigs with telomeric sequences on either the 5′ or 3′ end. A methylome database of the genome was constructed using SMRT and m4C and m6A methylated nucleotides, and many unknown modification types were identified. The major m6A methylation motif is GA and GGAG, and the major m4C methylation…
Background Helicobacter pylori is a Gram-negative bacterium which mainly causes peptic ulcer disease in human, but is also the predominant cause of stomach cancer. It has been coevolving with human since 120,000 years and, according to Multi-locus sequence typing (MLST), H. pylori can be classified into seven major population types, namely, hpAfrica1, hpAfrica2, hpNEAfrica, hpEastAsia, hpAsia2, hpEurope and hpSahul. Helicobacter pylori harbours a large number of restriction-modification (R-M) systems. The methyltransferase (MTase) unit plays a significant role in gene regulation and also possibly modulates pathogenicity. The diversity in MTase can act as geomarkers to correlate strains with the phylogeographic origins.…