Obtaining microbial genomes with the highest accuracy and contiguity is extremely important when exploring the functional impact of genetic and epigenetic variants on a genome-wide scale. A comprehensive view of the bacterial genome, including genes, regulatory regions, IS elements, phage integration sites, and base modifications is vital to understanding key traits such as antibiotic resistance, virulence, and metabolism. SMRT Sequencing provides complete genomes, often assembled into a single contig. Our streamlined microbial multiplexing procedure for the Sequel System, from library preparation to genome assembly, can be completed with less than 8 hours bench time. Starting with high-quality genomic DNA (gDNA),…
Our understanding of microbiology has evolved enormously over the last 150 years. Few institutions have witnessed our collective progress more closely than the National Collection of Type Cultures (NCTC). In fact, the collection itself is a record of the many milestones microbiologists have crossed, building on the discoveries of those who came before. To date, 60% of NCTC’s historic collection now has a closed, finished reference genome, thanks to PacBio Single Molecule, Real- Time (SMRT) Sequencing. We are excited to be their partner in crossing this latest milestone on their quest to improve human and animal health by understanding the…
The UK’s National Collection of Type Cultures (NCTC) is a unique collection of more than 5,000 expertly preserved and authenticated bacterial cultures, many of historical significance. Founded in 1920, NCTC is the longest established collection of its type anywhere in the world, with a history of its own that has reflected — and contributed to — the evolution of microbiology for more than 100 years.
Highly accurate long reads – HiFi reads – with single-molecule resolution make Single Molecule, Real-Time (SMRT) Sequencing ideal for full-length 16S rRNA sequencing, shotgun metagenomic profiling, and metagenome 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.
One of the major applications of DNA sequencing technology is to bring together information that is distant in sequence space so that understanding genome structure and function becomes easier on a large scale. The Single Molecule Real Time (SMRT) Sequencing platform provides direct sequencing data that can span several thousand bases to tens of thousands of bases in a high-throughput fashion. In contrast to solving genomic puzzles by patching together smaller piece of information, long sequence reads can decrease potential computation complexity by reducing combinatorial factors significantly. We demonstrate algorithmic approaches to construct accurate consensus when the differences between reads…
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…
The newer hierarchical genome assembly process (HGAP) performs de novo assembly using data from a single PacBio long insert library. To assess the benefits of this method, DNA from several Salmonella enterica serovars was isolated from a pure culture. Genome sequencing was performed using Pacific Biosciences RS sequencing technology. The HGAP process enabled us to close sixteen Salmonella subsp. enterica genomes and their associated mobile elements: The ten serotypes include: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) S. Bareilly, S. Heidelberg, S. Cubana, S. Javiana and S. Typhimurium, S. Newport, S. Montevideo, S. Agona, and S. Tennessee. In addition,…
Background: Microbial ecology is reshaping our understanding of the natural world by revealing the large phylogenetic and functional diversity of microbial life. However the vast majority of these microorganisms remain poorly understood, as most cultivated representatives belong to just four phylogenetic groups and more than half of all identified phyla remain uncultivated. Characterization of this microbial ‘dark matter’ will thus greatly benefit from new metagenomic methods for in situ analysis. For example, sensitive high throughput methods for the characterization of community composition and structure from the sequencing of conserved marker genes. Methods: Here we utilize Single Molecule Real-Time (SMRT) sequencing…
While the utility of Single Molecule, Real-Time (SMRT) Sequencing for de novo assembly and finishing of bacterial isolates is well established, this technology has not yet been widely applied to shotgun sequencing of microbial communities. In order to demonstrate the feasibility of this approach, we sequenced genomic DNA from the Microbial Mock Community B of the Human Microbiome Project
Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers to understand molecular mechanisms in evolution and gain insight into adaptive strategies. With read lengths exceeding 10 kb, we are able to sequence high-quality, closed microbial genomes with associated plasmids, and investigate large genome complexities, such as long, highly repetitive, low-complexity regions and multiple tandem-duplication events. Improved genome quality, observed at 99.9999% (QV60) consensus accuracy, and significant reduction of gap regions in reference genomes (up to and beyond 50%) allow researchers to better understand coding sequences with high confidence, investigate potential regulatory mechanisms in noncoding regions, and make inferences…
The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. One popular, efficient method of profiling communities is to amplify and sequence the evolutionarily conserved 16S rRNA sequence. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost and species-level classification is often not possible. SMRT Sequencing easily spans the entire 1.5 kb 16S gene, and in combination with highly-accurate single-molecule sequences, can improve the identification of individual species in a metapopulation. However, when amplifying…
The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. Amplification and sequencing of the evolutionarily conserved 16S rRNA gene is an efficient method of profiling communities. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost, and species-level classification is often not possible. PacBio SMRT Sequencing easily spans the entire 1.5 kb 16S gene in a single read, producing highly accurate single-molecule sequences that can improve the identification of individual species in a metapopulation.However,…
As the throughput of the PacBio Systems continues to increase, so has the desire to fully utilize SMRT Cell sequencing capacity to multiplex microbes for whole genome sequencing. Multiplexing is readily achieved by incorporating a unique barcode for each microbe into the SMRTbell adapters and using a streamlined library preparation process. Incorporating barcodes without PCR amplification prevents the loss of epigenetic information and the generation of chimeric sequences, while eliminating the need to generate separate SMRTbell libraries. We multiplexed the genomes of up to 8 unique strains of H. pylori. Each genome was sheared and processed through adapter ligation in…
Background: The Nanobind technology from Circulomics provides an elegant HMW DNA extraction solution for genome sequencing of Gram-positive and -negative microbes. Nanobind is a nanostructured magnetic disk that can be used for rapid extraction of high molecular weight (HMW) DNA from diverse sample types including cultured cells, blood, plant nuclei, and bacteria. Processing can be completed in 7 kb repeats. Fragment size was increased to ~14 kb, with some fragments >30 kb. Results: Here we present a demonstration of these capabilities using isolates relevant to high-throughput sequencing applications, including common foodborne pathogens (Shigella, Listeria, Salmonella), and species often seen in…