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Tuesday, June 1, 2021

Long-read, single-molecule applications for protein engineering.

The long read lengths of PacBio’s SMRT Sequencing enable detection of linked mutations across multiple kilobases of sequence. This feature is particularly useful in the context of protein engineering, where large numbers of similar constructs are generated routinely to explore the effects of mutations on function and stability. We have developed a PCR-based barcoded sequencing method to generate high quality, full-length sequence data for batches of constructs generated in a common backbone. Individual barcodes are coupled to primers targeting a common region of the vector of interest. The amplified products are pooled into a single DNA library, and sequencing data…

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Tuesday, June 1, 2021

Accurately surveying uncultured microbial species with SMRT Sequencing

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…

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Tuesday, June 1, 2021

SMRT Sequencing and assembly of the human microbiome project Mock Community sample – a feasibility project.

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

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Tuesday, June 1, 2021

An interactive workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data.

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…

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Tuesday, June 1, 2021

Developments in PacBio metagenome sequencing: Shotgun whole genomes and full-length 16S.

The assembly of metagenomes is dramatically improved by the long read lengths of SMRT Sequencing. This is demonstrated in an experimental design to sequence a mock community from the Human Microbiome Project, and assemble the data using the hierarchical genome assembly process (HGAP) at Pacific Biosciences. Results of this analysis are promising, and display much improved contiguity in the assembly of the mock community as compared to publicly available short-read data sets and assemblies. Additionally, the use of base modification information to make further associations between contigs provides additional data to improve assemblies, and to distinguish between members within a…

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Tuesday, June 1, 2021

SMRT Sequencing solutions for investigative studies to understand evolutionary processes.

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…

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Tuesday, June 1, 2021

Genome analysis of a bacterium that causes lameness.

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…

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Tuesday, June 1, 2021

Comparative genome analysis of Clavibacter michiganensis subsp. michiganensis strains provides insights into genetic diversity and virulence.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram positive actinomycete, causing bacterial canker of tomato (Solanum lycopersicum) a disease that can cause significant losses in tomato production. In this study, we determined the complete genome sequence of 13 California Cmm strains and one saprophytic Clavibacter strain using a combination of Ilumina and PacBio sequencing. The California Cmm strains have genome size (3.2 -3.3 mb) similar to the reference strain NCPPB382 (3.3 mb) with =98% sequence identity. Cmm strains from California share =92% genes (8-10% are noble genes) with the reference Cmm strain NCPPB382. Despite this similarity, we detected significant alternatives…

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Tuesday, June 1, 2021

A genome assembly of the domestic goat from 70x coverage of single molecule, real-time sequence.

Goat is an important source of milk, meat, and fiber, especially in developing countries. An advantage of goats as livestock is the low maintenance requirements and high adaptability compared to other milk producers. The global population of domestic goats exceeds 800 million. In Africa, goat production is characterized by low productivity levels, and attempts to introduce more productive breeds have met with poor success due in part to nutritional constraints. It has been suggested that incorporation of selective breeding within the herds adapted for survival could represent one approach to improving food security across Africa. A recently produced genome assembly…

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Tuesday, June 1, 2021

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence,…

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Tuesday, June 1, 2021

Low-input long-read sequencing for complete microbial genomes and metagenomic community analysis.

Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. A 2 kb SMRTbell library only requires a few ng of gDNA when carrier DNA is added to the library. The resulting libraries can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes…

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Tuesday, June 1, 2021

Metagenomes of native and electrode-enriched microbial communities from the Soudan Iron Mine.

Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan Underground Iron Mine harbor active microbial communities. To characterize these assemblages, we performed shotgun metagenomics of native and enriched samples. Following enrichment on poised electrodes and long read sequencing, we recovered from the metagenome the closed, circular genome of a novel Desulfuromonas sp. with remarkable genomic features that were not fully resolved by short read assembly alone. This organism was essentially absent in unenriched Soudan communities, indicating that electrodes are highly selective for putative metal reducers. Native community metagenomes suggest that carbon cycling is driven by methyl-C1 metabolism, in…

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Tuesday, June 1, 2021

Barcoding strategies for multiplexing of samples using a long-read sequencing technology.

We have developed barcoding reagents and workflows for multiplexing amplicons or fragmented native genomic (DNA) prior to Single Molecule, Real-Time (SMRT) Sequencing. The long reads of PacBio’s SMRT Sequencing enable detection of linked mutations across multiple kilobases (kb) of sequence. This feature is particularly useful in the context of mutational analysis or SNP confirmation, where a large number of samples are generated routinely. To validate this workflow, a set of 384 1.7-kb amplicons, each derived from variants of the Phi29 DNA polymerase gene, were barcoded during amplification, pooled, and sequenced on a single SMRT Cell. To demonstrate the applicability of…

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