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Sunday, September 22, 2019

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing,…

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Sunday, September 22, 2019

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic…

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Sunday, September 22, 2019

Koumiss consumption alleviates symptoms of patients with chronic atrophic gastritis: A possible link To modulation of gut microbiota

Intestinal dysbiosisis closely related to a variety of medical conditions, especially gastrointestinal diseases. The present study aimed to investigate the effects of koumiss on chronic atrophic gastritis (CAG) in an out-patient clinical trial (n = 10; all female subjects aged 41-55; body mass index ranging from 19.5 to 25.8). Each patient consumed three servings of koumiss per day (i.e. 250 ml daily before each of 3 meals) for a 60-day period. The improvement of patients’ symptoms was monitored by comparing the total scores of symptoms before and after the treatment. Meanwhile, the changes in the patients’ fecal microbiota composition and…

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Sunday, September 22, 2019

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT…

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Sunday, September 22, 2019

Interaction between the microbiome and TP53 in human lung cancer.

Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort.Overall, we demonstrate a lower alpha diversity in normal…

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Sunday, September 22, 2019

A single-cell genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including…

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Sunday, September 22, 2019

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its…

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Sunday, September 22, 2019

Mutations in genes encoding Penicillin-binding proteins and efflux pumps play a role in ß-lactam resistance in Helicobacter cinaedi.

ß-Lactams are often used to treatHelicobacter cinaediinfections; however, the mechanism underlying ß-lactam resistance is unknown. In this study, we investigated ß-lactam resistance in anH. cinaedistrain, MRY12-0051 (MICs of amoxicillin [AMX] and ceftriaxone [CRO], 32 and 128 µg/ml; obtained from human feces). Based on a comparative whole-genome analysis of MRY12-0051 and the CRO-susceptibleH. cinaedistrain MRY08-1234 (MICs of AMX and CRO, 1 and 4 µg/ml; obtained from human blood), we identified five mutations in genes encoding penicillin-binding proteins (PBPs), including two inpbpA, one inpbp2, and two inftsITransformation and penicillin binding assays indicated that CRO resistance was mainly associated with mutations inpbpA; mutations…

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Sunday, September 22, 2019

Basic characterization of natural transformation in a highly transformable Haemophilus parasuis strain SC1401.

Haemophilus parasuis causes Glässer’s disease and pneumonia, incurring serious economic losses in the porcine industry. In this study, natural competence was investigated in H. parasuis. We found competence genes in H. parasuis homologous to ones in Haemophilus influenzae and a high consensus battery of Sxy-dependent cyclic AMP (cAMP) receptor protein (CRP-S) regulons using bioinformatics. High rates of natural competence were found from the onset of stationary-phase growth condition to mid-stationary phase (OD600 from 0.29 to 1.735); this rapidly dropped off as cells reached mid-stationary phase (OD600 from 1.735 to 1.625). As a whole, bacteria cultured in liquid media were observed…

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Sunday, September 22, 2019

N4-cytosine DNA methylation regulates transcription and pathogenesis in Helicobacter pylori.

Many bacterial genomes exclusively display an N4-methyl cytosine base (m4C), whose physiological significance is not yet clear. Helicobacter pylori is a carcinogenic bacterium and the leading cause of gastric cancer in humans. Helicobacter pylori strain 26695 harbors a single m4C cytosine methyltransferase, M2.HpyAII which recognizes 5′ TCTTC 3′ sequence and methylates the first cytosine residue. To understand the role of m4C modification, M2.hpyAII deletion strain was constructed. Deletion strain displayed lower adherence to host AGS cells and reduced potential to induce inflammation and apoptosis. M2.hpyAII gene deletion strain exhibited reduced capacity for natural transformation, which was rescued in the complemented…

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Sunday, September 22, 2019

A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions.

Many bacteria utilize simple DNA sequence repeats as a mechanism to randomly switch genes on and off. This process is called phase variation. Several phase-variable N6-adenine DNA-methyltransferases from Type III restriction-modification systems have been reported in bacterial pathogens. Random switching of DNA methyltransferases changes the global DNA methylation pattern, leading to changes in gene expression. These epigenetic regulatory systems are called phasevarions – phase-variable regulons. The extent of these phase-variable genes in the bacterial kingdom is unknown. Here, we interrogated a database of restriction-modification systems, REBASE, by searching for all simple DNA sequence repeats in mod genes that encode Type III N6-adenine…

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Sunday, September 22, 2019

CagY-dependent regulation of type IV secretion in Helicobacter pylori is associated with alterations in integrin binding.

Strains of Helicobacter pylori that cause ulcer or gastric cancer typically express a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI). CagY is an ortholog of VirB10 that, unlike other VirB10 orthologs, has a large middle repeat region (MRR) with extensive repetitive sequence motifs, which undergo CD4+ T cell-dependent recombination during infection of mice. Recombination in the CagY MRR reduces T4SS function, diminishes the host inflammatory response, and enables the bacteria to colonize at a higher density. Since CagY is known to bind human a5ß1 integrin, we tested the hypothesis that recombination in the CagY MRR…

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Sunday, September 22, 2019

Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing.

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate…

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Sunday, September 22, 2019

Complete genome sequencing and comparative genomic analysis of Helicobacter apodemus isolated from the wild Korean striped field mouse (Apodemus agrarius) for potential pathogenicity

The Helicobacter bacterial genus comprises of spiral-shaped gram-negative bacteria with flagella that colonize the gastro-intestinal (GI) tract of humans and various mammals (Solnick and Schauer, 2001). In particular, Helicobacter pylori was classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) in 1994, and has been shown to occur with a high prevalence in humans, although this varies between geographical regions, ethnic groups, and various populations (Kusters et al., 2006; Goh et al., 2011). To date, more than 37 Helicobacter species have been identified in addition to H. pylori (Péré-Védrenne et al., 2017). Furthermore, non-H.…

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Sunday, September 22, 2019

A novel probiotic, Lactobacillus johnsonii 456, resists acid and can persist in the human gut beyond the initial ingestion period.

Probiotics are considered to have multiple beneficial effects on the human gastrointestinal tract, including immunomodulation, pathogen inhibition, and improved host nutrient metabolism. However, extensive characterization of these properties is needed to define suitable clinical applications for probiotic candidates. Lactobacillus johnsonii 456 (LBJ 456) was previously demonstrated to have anti-inflammatory and anti-genotoxic effects in a mouse model. Here, we characterize its resistance to gastric and bile acids as well as its ability to inhibit gut pathogens and adhere to host mucosa. While bile resistance and in vitro host attachment properties of LBJ 456 were comparable to other tested probiotics, LBJ 456…

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