June 1, 2021  |  

SMRT Sequencing of whole mitochondrial genomes and its utility in association studies of metabolic disease.

In this study we demonstrate the utility of Single-Molecule Real Time SMRT sequencing to detect variants and to recapitulate whole mitochondrial genomes in an association study of Metabolic syndrome using samples from a well-studied cohort from Micronesia. The Micronesian island of Kosrae is a rare genetic isolate that offers significant advantages for genetic studies of human disease. Kosrae suffers from one of the highest rates of MetS (41%), obesity (52%), and diabetes (17%) globally and has a homogeneous environment making this an excellent population in which to study these significant health problems. We are conducting family-based association analyses aimed at identifying specific mitochondrial variants that contribute to obesity and other co-morbid conditions. We sequenced whole mitochondrial genomes from 10 Kosraen individuals who represent greater than 25 % of the mitochondrial genetic diversity for the entire Kosraen population. Using Pacific Biosciences C2 chemistry, SMRTbell libraries were constructed from pooled, full-length, unsheared 5 kb PCR amplicons, tiling the entire 16.6 kb mtDNA genome. Average read lengths for each sample were between 2500-3000 bp, with 5% of reads between 6,000-8,000 bases, depending on movie lengths. The data generated in this study serve as proof of principle that SMRT Sequencing data can be utilized for identification of high-quality variants and complete mitochondrial genome sequences. These data will be leveraged to identify causative variants for Metabolic syndrome and associated disorders.


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.


June 1, 2021  |  

De novo assembly of a complex panicoid grass genome using ultra-long PacBio reads with P6C4 chemistry

Drought is responsible for much of the global losses in crop yields and understanding how plants naturally cope with drought stress is essential for breeding and engineering crops for the changing climate. Resurrection plants desiccate to complete dryness during times of drought, then “come back to life” once water is available making them an excellent model for studying drought tolerance. Understanding the molecular networks governing how resurrection plants handle desiccation will provide targets for crop engineering. Oropetium thomaeum (Oro) is a resurrection plant that also has the smallest known grass genome at 250 Mb compared to Brachypodium distachyon (300 Mb) and rice (350 Mb). Plant genomes, especially grasses, have complex repeat structures such as telomeres, centromeres, and ribosomal gene cassettes, and high heterozygosity, which makes them difficult to assembly using short read next generation sequencing technologies. Ultra-long PacBio reads using the new P6C4 chemistry and the latest 15kb Blue Pippin size-selection protocol to generate 20kb insert libraries that yielded an average read length of 12kb providing ~72X coverage, and 10X coverage with reads over 20kb. The HGAP assembly covers 98% of the genome with a contig N50 of 2.4 Mb, which makes it one of the highest quality and most complete plant genomes assembled to date. Oro has a compact genome structure compared to other grasses with only 16% repeat sequences but has very good collinearity with other grasses. Understanding the genomic mechanisms of extreme desiccation tolerance in resurrection plants like Oro will provide insights for engineering and intelligent breeding of improved food, fuel, and fiber crops.


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.


June 1, 2021  |  

WGS SMRT Sequencing of patient samples from a fecal microbiota transplant trial

Fecal samples were obtained from human subjects in the first blinded, placebo-controlled trial to evaluate the efficacy and safety of fecal microbiota transplant (FMT) for treatment of recurrent C. difficile infection. Samples included pre-and post-FMT transplant, post-placebo transplant, and the donor control; samples were taken at 2 and 8 week post-FMT. Sequencing was done on the PacBio Sequel System, with the goal of obtaining high quality sequences covering whole genes or gene clusters, which will be used to better understand the relationship between the composition and functional capabilities of intestinal microbiomes and patient health. Methods: Samples were randomly sheared to 2-3 kb fragments, a sufficient length to cover most genes, and SMRTbell libraries were prepared using standard protocols. Libraries were run on the Sequel System, which has a throughput of hundreds of thousands of reads per SMRT Cell, adequate yield to sample the complex microbiomes of post-transplant and donor samples.Results: Here we characterize samples, describe library prep methods and detail Sequel System operation, including run conditions. Descriptive statistics of data output (primary analysis) are presented, along with SMRT Analysis reports on circular consensus sequence (CCS) reads generated using an updated algorithm (CCS2). Final sequencing yields are filtered at various levels of predicted accuracy from 90% to 99.9%. Previous studies done using the PacBio RS II System demonstrated the ability to profile at the species level, and in some cases the strain level, and provided functional insight. Conclusions: These results demonstrate that the Sequel System is well-suited for characterization of complex microbial communities, with the ability for high-throughput generation of extremely accurate single-molecule sequences, each several kilobases in length. The entire process from shearing and library prep through sequencing and CCS analysis can be completed in less than 48 hours.


June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

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 new, high throughput data type uniquely suited to metagenome characterization: HiFi reads. HiFi reads combine high accuracy with read lengths up to 15 kb, eliminating the need for assembly for most microbiome applications, including functional profiling, gene discovery, and metabolic pathway reconstruction. Here we present the application of the HiFi data type to enable a new method of analyzing metagenomes that does not require assembly.


June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

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 addresses these challenges: HiFi reads. HiFi reads combine QV20 accuracy with long read lengths, eliminating the need for assembly for most metagenome applications, including gene discovery and metabolic pathway reconstruction. In fact, the read lengths and accuracy of HiFi data match or outperform the quality metrics of most metagenome assemblies, enabling cost-effective recovery of intact genes and operons while omitting the resource intensive and data-inefficient assembly step. Here we present the application of HiFi sequencing to both mock and human fecal samples using full-length 16S and shotgun methods. This proof-of-concept work demonstrates the unique strengths of the HiFi method. First, the high correspondence between the expected community composition,16S and shotgun profiling data reflects low context bias. In addition, every HiFi read yields ~5-8 predicted genes, without assembly, using standard tools. If assembly is desired, excellent results can be achieved with Canu and contig binning tools. In summary, HiFi sequencing is a new, cost-effective option for high-resolution functional profiling of metagenomes which complements existing short read workflows.


June 1, 2021  |  

Comparative metagenome-assembled genome analysis of “Candidatus Lachnocurva vaginae”, formerly known as Bacterial Vaginosis Associated bacterium – 1 (BVAB1)

Bacterial Vaginosis Associated bacterium 1 (BVAB1) is an as-yet uncultured bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales. As its name suggests, this bacterium is often associated with bacterial vaginosis (BV), a common vaginal disorder that has been shown to increase a woman’s risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae infections as well as preterm birth. Further, BVAB1 is associated with the persistence of BV following metronidazole treatment, increased vaginal inflammation, and adverse obstetrics outcomes. There is no available complete genome sequence of BVAB1, which has made it di?cult to mechanistically understand its role in disease. We present here a circularized metagenome-assembled genome (cMAG) of B VAB1 as well as a comparative analysis including an additional six metagenome-assembled genomes (MAGs) of this species. These sequences were derived from cervicovaginal samples of seven separate women. The cMAG is 1.649 Mb in size and encodes 1,578 genes. We propose to rename BVAB1 to “Candidatus Lachnocurva vaginae” based on phylogenetic analyses, and provide genomic evidence that this candidate species may metabolize D-lactate, produce trimethylamine (one of the chemicals responsible for BV-associated odor), and be motile. The cMAG and the six MAGs are valuable resources that will further contribute to our understanding of the heterogeneous etiology of bacterial vaginosis.


June 1, 2021  |  

Metagenomic analysis of type II diabetes gut microbiota using PacBio HiFi reads reveals taxonomic and functional differences

In the past decade, the human microbiome has been increasingly shown to play a major role in health. For example, imbalances in gut microbiota appear to be associated with Type II diabetes mellitus (T2DM) and cardiovascular disease. Coronary artery disease (CAD) is a major determinant of the long-term prognosis among T2DM patients, with a 2- to 4-fold increased mortality risk when present. However, the exact microbial strains or functions implicated in disease need further investigation. From a large study with 523 participants (185 healthy controls, 186 T2DM patients without CAD, and 106 T2DM patients with CAD), 3 samples from each patient group were selected for long read sequencing. Each sample was prepared and sequenced on one Sequel II System SMRT Cell, to assess whether long accurate PacBio HiFi reads could yield additional insights to those made using short reads. Each of the 9 samples was subject to metagenomic assembly and binning, taxonomic classification and functional profiling. Results from metagenomic assembly and binning show that it is possible to generate a significant number of complete MAGs (Metagenome Assembled Genomes) from each sample, with over half of the high-quality MAGs being represented by a single circular contig. We show that differences found in taxonomic and functional profiles of healthy versus diabetic patients in the small 9-sample study align with the results of the larger study, as well as with results reported in literature. For example, the abundances of beneficial short- chain fatty acid (SCFA) producers such as Phascolarctobacterium faecium and Faecalibacterium prausnitzii were decreased in T2DM gut microbiota in both studies, while the abundances of quinol and quinone biosynthesis pathways were increased as compared to healthy controls. In conclusion, metagenomic analysis of long accurate HiFi reads revealed important taxonomic and functional differences in T2DM versus healthy gut microbiota. Furthermore, metagenome assembly of long HiFi reads led to the recovery of many complete MAGs and a significant number of complete circular bacterial chromosome sequences.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.