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June 1, 2021  |  

Getting the most out of your PacBio libraries with size selection.

PacBio RS II sequencing chemistries provide read lengths beyond 20 kb with high consensus accuracy. The long read lengths of P4-C2 chemistry and demonstrated consensus accuracy of 99.999% are ideal for applications such as de novo assembly, targeted sequencing and isoform sequencing. The recently launched P5-C3 chemistry generates even longer reads with N50 often >10,000 bp, making it the best choice for scaffolding and spanning structural rearrangements. With these chemistry advances, PacBio’s read length performance is now primarily determined by the SMRTbell library itself. Size selection of a high-quality, sheared 20 kb library using the BluePippin™ System has been demonstrated to increase the N50 read length by as much as 5 kb with C3 chemistry. BluePippin size selection or a more stringent AMPure® PB selection cutoff can be used to recover long fragments from degraded genomic material. The selection of chemistries, P4-C2 versus P5-C3, is highly dependent on the final size distribution of the SMRTbell library and experimental goals. PacBio’s long read lengths also allow for the sequencing of full-length cDNA libraries at single-molecule resolution. However, longer transcripts are difficult to detect due to lower abundance, amplification bias, and preferential loading of smaller SMRTbell constructs. Without size selection, most sequenced transcripts are 1-1.5 kb. Size selection dramatically increases the number of transcripts >1.5 kb, and is essential for >3 kb transcripts.

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June 1, 2021  |  

Using whole exome sequencing and bacterial pathogen sequencing to investigate the genetic basis of pulmonary non-tuberculous mycobacterial infections.

Pulmonary non-tuberculous mycobacterial (PNTM) infections occur in patients with chronic lung disease, but also in a distinct group of elderly women without lung defects who share a common body morphology: tall and lean with scoliosis, pectus excavatum, and mitral valve prolapse. In order to characterize the human host susceptibility to PNTM, we performed whole exome sequencing (WES) of 44 individuals in extended families of patients with active PNTM as well as 55 additional unrelated individuals with PNTM. This unique collection of familial cohorts in PNTM represents an important opportunity for a high yield search for genes that regulate mucosal immunity. An average of 58 million 100bp paired-end Illumina reads per exome were generated and mapped to the hg19 reference genome. Following variant detection and classification, we identified 58,422 potentially high-impact SNPs, 97.3% of which were missense mutations. Segregating variants using the family pedigrees as well as comparisons to the unrelated individuals identified multiple potential variants associated with PNTM. Validations of these candidate variants in a larger PNTM cohort are underway. In addition to WES, we sequenced the genomes of 52 mycobacterial isolates, including 9 from these PNTM patients, to integrate host PNTM susceptibility with mycobacterial genotypes and gain insights into the key factors involved in this devastating disease. These genomes were sequenced using a combination of 454, Illumina, and PacBio platforms and assembled using multiple genome assemblers. The resulting genome sequences were used to identify mycobacterial genotypes associated with virulence, invasion, and drug resistance.

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June 1, 2021  |  

New discoveries from closing Salmonella genomes using Pacific Biosciences continuous long reads.

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, we were able to detect novel methyltransferases (MTases) by using the Pacific Biosciences kinetic score distributions showing that each serovar appears to have a novel methylation pattern. For example while all Salmonella serovars examined so far have methylase specific activity for 5’-GATC-3’/3’-CTAG-5’ and 5’-CAGAG-3’/3’-GTCTC-5’ (underlined base indicates a modification), S. Heidelberg is uniquely specific for 5’-ACCANCC-3’/3’-TGGTNGG-5’, while S. Typhimurium has uniquely methylase specific for 5′-GATCAG-3’/3′- CTAGTC-5′ sites, for the samples examined so far. We believe that this may be due to the unique environments and phages that these serotypes have been exposed to. Furthermore, our analysis identified and closed a variety of plasmids such as mobilization plasmids, antimicrobial resistance plasmids and IncX plasmids carrying a Type IV secretion system (T4SS). The VirB/D4 T4SS apparatus is important in that it assists with rapid dissemination of antibiotic resistance and virulence determinants. Presently, only limited information exists regarding the genotypic characterization of drug resistance in S. Heidelberg isolates derived from various host species. Here, we characterize two S. Heidelberg outbreak isolates from two different outbreaks. Both isolates contain the IncX plasmid of approximately 35 kb, and carried the genes virB1, virB2, virB3/4, virB5, virB6, virB7, virB8, virB9, virB10, virB11, virD2, and virD4, that are associated with the T4SS. In addition, the outbreak isolate associated with ground turkey carries a 4,473 bp mobilization plasmid and an incompatibility group (Inc) I1 antimicrobial resistance plasmid encoding resistance to gentamicin (aacC2), beta-lactam (bl2b_tem), streptomycin (aadAI) and tetracycline (tetA, tetR) while the outbreak isolate associated with chicken breast carries the IncI1 plasmid encoding resistance to gentamicin (aacC2), streptomycin (aadAI) and sulfisoxazole (sul1). Using this new technology we explored the genetic elements present in resistant pathogens which will achieve a better understanding of the evolution of Salmonella.

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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 of full-length 16S rRNA amplicons to phylogenetically profile microbial communities to below the genus-level. We test this method on a mock community of known composition, as well as a previously studied microbial community from a lake known to predominantly contain poorly characterized phyla. These results are compared to traditional 16S tag sequencing from short-read technologies and subsets of the full-length data corresponding to the same regions of the 16S gene. Results: We explore the benefits of using full-length amplicons for estimating community structure and diversity. In addition, we investigate the possible effects of context-specific and GC-content biases known to affect short-read sequencing technologies on the predicted community structure. We characterize the potential benefits of profiling metagenomic communities with full-length 16S rRNA genes from SMRT sequencing relative to standard methods.

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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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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 present a collection of tools tailored for PacBio data for the analysis of these fragmented metagenomic assembles, allowing improvements in the assembly results, and greater insight into the communities themselves. Supervised classification is applied to a large set of sequence characteristics, e.g., GC content, raw-read coverage, k-mer frequency, and gene prediction information, allowing the clustering of contigs from single or highly related species. A unique feature of SMRT Sequencing data is the availability of base modification / methylation information, which can be used to further analyze clustered contigs expected to be comprised of single or very closely related species. Here we show base modification information can be used to further study variation, based on differences in the methylated DNA motifs involved in the restriction modification system. Application of these techniques is demonstrated on a monkey intestinal microbiome sample and an in silico mix of real sequencing data from distinct bacterial samples.

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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 microbial community. The epigenetic approach is a novel validation method unique to SMRT Sequencing. In addition to whole-genome shotgun sequencing, SMRT Sequencing also offers improved classification resolution and reliability of metagenomic and microbiome samples by the full-length sequencing of 16S rRNA (~1500 bases long). Microbial communities can be detected at the species level in some cases, rather than being limited to the genus taxonomic classification as constrained by short-read technologies. The performance of SMRT Sequencing for these metagenomic samples achieved >99% predicted concordance to reference sequences in cecum, soil, water, and mock control investigations for bacterial 16S. Community samples are estimated to contain from 2.3 and up to 15 times as many species with abundance levels as low as 0.05% compared to the identification of phyla groups.

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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 BCO three-fold when compared with broilers drinking tap water (P = 0.001). We found that the minimum effective dose of Staphylococcus agnetis to induce BCO in broilers grown on wire flooring experiment is 105 cfu/ml. We used PacBio and Illumina sequencing to assemble a 2.4 Mbp contig representing the genome and a 34 kbp contig for the largest plasmid of S. agnetis. Annotation of this genome is underway through comparative genomics with other Staphylococcus genomes, and identification of virulence factors. Our goal is to elucidate genetic diversity, toxins, and pathogenicity determinants, for this poorly characterized species. Isolating pathogenic bacterial species, defining their likely route of transmission to broilers, and genomic analyses will contribute substantially to the development of measures for mitigating BCO losses in poultry.

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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 in California strains with respect to plasmid number, plasmid composition, and genomic island presence indicating acquisition of unique mechanisms controlling virulence. Plasmids pCM1 and pCM2, that were previously demonstrated to be required for NCPPB382 virulence, also differ in their presence and gene content across Cmm strains. pCM2 is absent in some Cmm strains and that still retain virulence in tomato. Saprophytic Clavibacter possess a novel plasmid, pSCM, and lacks the majority of characterized virulence factors. Genome sequence information was also used to design specific and sensitive primer pairs for Cmm detection. A mechanistic understanding of how genomic changes have impacted Cmm virulence and survival across diverse strains will be necessary for developing a robust disease control strategies for bacterial canker of tomato.

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June 1, 2021  |  

A workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data

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 the analysis of PacBio metagenomic assemblies. These tools allow for improvements in the assembly results, and greater insight into the complexity of the study communities. Supervised classification is applied to a large set of sequence characteristics (e.g. GC content, raw read coverage, k-mer frequency, and gene prediction information) and to cluster contigs from single or highly related species. Assembly in isolation of the raw data associated with these contigs is shown to improve assembly statistics. A unique feature of SMRT Sequencing is the availability to leverage simultaneously collected base modification / methylation data to aid the clustering of contigs expected to comprise a single or very closely related species. We demonstrate the added value of base modification information to distinguish and study variation within metagenomic samples based on differences in the methylated DNA motifs involved in the restriction modification system. Application of these techniques is demonstrated on a mock community and monkey intestinal microbiome sample.

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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, DNA methylation is characterized in the process of sequencing. PacBio® sequencing has also been applied to microbial transcriptomes. Long reads enable sequencing of full-length cDNAs allowing for identification of complete gene and operon sequences without the need for transcript assembly. We will highlight several examples where these capabilities have been leveraged in the areas of industrial microbiology, including biocommodities, biofuels, bioremediation, new bacteria with potential commercial applications, antibiotic discovery, and livestock/plant microbiome interactions.

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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 using the SMRT Portal assembly program HGAP, plus base-modification analysis. The entire process can be done in less than 3 days by standard laboratory personnel. This approach is particularly important for the analysis of metagenomic communities, in which genomic DNA is often limited. From these samples, full-length 16S amplicons can be generated, prepped with the standard SMRTbell library prep protocol, and sequenced. Alternatively, a 2 kb sheared library, made from a few ng of input DNA, can also be used to elucidate the microbial composition of a community, and may provide information about biochemical pathways present in the sample. In both these cases, 1-2 kb reads with >99% accuracy can be obtained from Circular Consensus Sequencing.

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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 particular methylotrophic methanogenesis. Our results highlight the promising potential for long reads in metagenomic surveys of low-diversity environments.

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June 1, 2021  |  

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing.

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments generally use short-read, second-generation sequencing, which results in data processing difficulties. For example, reads less than 1 kb in length will likely not cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, single molecule, real-time (SMRT) Sequencing reads in the 1-2 kb range, with >99% accuracy can be efficiently generated for low amounts of input DNA. 10 ng of input DNA sequenced in 4 SMRT Cells would generate >100,000 such reads. While throughput is low compared to second-generation sequencing, the reads are a true random sampling of the underlying community, since SMRT Sequencing has been shown to have no sequence-context bias. Long read lengths mean that that it would be reasonable to expect a high number of the reads to include gene fragments useful for analysis.

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