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
Lameness is a significant problem resulting in millions of dollars in lost revenue annually. In commercial broilers, the most common cause of lameness is bacterial chondronecrosis with osteomyelitis (BCO). We are using a wire flooring model to induce lameness attributable to BCO. We used 16S ribosomal DNA sequencing to determine that Staphylococcus spp. were the main species associated with BCO. Staphylococcus agnetis, which previously had not been isolated from poultry, was the principal species isolated from the majority of the bone lesion samples. Administering S. agnetis in the drinking water to broilers reared on wire flooring increased the incidence of…
Microbes play an important role in nearly every part of our world, as they affect human health, our environment, agriculture, and aid in waste management. Complete closed genome sequences, which have become the gold standard with PacBio long-read sequencing, can be key to understanding microbial functional characteristics. However, input requirements, consumables costs, and the labor required to prepare and sequence a microbial genome have in the past put PacBio sequencing out of reach for some larger projects. We have developed a multiplexed library prep approach that is simple, fast, and cost-effective, and can produce 4 to 16 closed bacterial genomes…
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…
We have streamlined the SMRTbell library generation protocols with improved workflows to deliver seamless end-to-end solutions from sample to analysis. A key improvement is the development of a single-tube reaction strategy that shortened hands-on time needed to generate each SMRTbell library, reduced time-consuming AM Pure purification steps, and minimized sample-handling induced gDNA damage to improve the integrity of long-insert SMRTbell templates for sequencing. The improved protocols support all large-insert genomic libraries, multiplexed microbial genomes, and amplicon sequencing. These advances enable completion of library preparation in less than a day (approximately 4 hours) and opens opportunities for automated library preparation for…
Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with short-read technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly requires high coverage for error correction, the benefits of unbiased coverage have in the past been lost for low abundance species. SMRT Sequencing performance improvements and the introduction of the Sequel II System has enabled a…
Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with one sequencing technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly typically requires high coverage for error correction, these benefits have in the past been lost for low-abundance species. The introduction of the Sequel II System has enabled a new, higher throughput, assembly-optional data type that…
Complete, high-quality microbial genomes are very valuable across a broad array of fields, from environmental studies, to human microbiome health, food pathogen surveillance, etc. Long-read sequencing enables accurate resolution of complex microbial genomes and is becoming the new standard. Here we report our novel Microbial Assembly pipeline to facilitate rapid, large-scale analysis of microbial genomes. We sequenced a 48-plex library with one SMRT Cell 8M on the Sequel II System, demultiplexed, then analyzed the data with Microbial Assembly.
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),…
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.
Interested to learn about pangenomes? Explore this guide to learn how they provide a more complete picture of the core genes of a given species and how that can provide better biological understanding.
In this PacBio User Group Meeting presentation, PacBio scientist Kristin Mars speaks about recent updates, such as the single-day library prep that’s now possible with the Iso-Seq Express workflow. She also notes that one SMRT Cell 8M is sufficient for most Iso-Seq experiments for whole transcriptome sequencing at an affordable price.
In this PacBio User Group Meeting presentation, PacBio scientist Meredith Ashby shared several examples of analysis — from full-length 16S sequencing to shotgun sequencing — showing how SMRT Sequencing enables accurate representation for metagenomics and microbiome characterization, in some cases even without fully assembling genomes. New updates will provide users with a dedicated microbial assembly pipeline, optimized for all classes of bacteria, as well as increased multiplexing on the Sequel II System, now with 48 validated barcoded adapters. That throughput could reduce the cost of microbial analysis substantially.
Understanding interactions among plants and the complex communities of organisms living on, in and around them requires more than one experimental approach. A new method for de novo metagenome assembly, PacBio HiFi sequencing, has unique strengths for determining the functional capacity of metagenomes. With HiFi sequencing, the accuracy and median read length of unassembled data outperforms the quality metrics for many existing assemblies generated with other technologies, enabling cost-competitive recovery of full-length genes and operons even from rare species. When paired with the ability to close the genomes of even challenging isolates like Xanthomonas, the PacBio Sequel II System is…
Microbial Assembly is our latest pipeline, specifically designed to assemble bacterial genomes (between 2 and 10 Mb) and plasmids. This pipeline includes the implementation of a new, circular-aware read alignment tool (Raptor), among other algorithmic improvements, which will be covered in this webinar. The topics covered include, staged assembly of bacterial chromosomes and plasmids, implementation of Raptor, a circular-aware read aligner, himeric read detection, origin of replication orientation, troubleshooting and more.