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  • Next Generation Sequencing (NGS)
    Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Xi Wang, Genes and Diseases - 2020
    Abstract
    Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5–10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.
    Topography of the respiratory tract bacterial microbiota in cattle
    Christopher McMullen, Microbiome - 2020
    Abstract
    Background Bacterial bronchopneumonia (BP) is the leading cause of morbidity and mortality in cattle. The nasopharynx is generally accepted as the primary source of pathogenic bacteria that cause BP. However, it has recently been shown in humans that the oropharynx may act as the primary reservoir for pathogens that reach the lung. The objective was therefore to describe the bacterial microbiota present along the entire cattle respiratory tract to determine which upper respiratory tract (URT) niches may contribute the most to the composition of the lung microbiota. Methods Seventeen upper and lower respiratory tract locations were sampled from 15 healthy feedlot steer calves. Samples were collected using a combination of swabs, protected specimen brushes, and saline washes. DNA was extracted from each sample and the 16S rRNA gene (V3-V4) was sequenced. Community composition, alpha-diversity, and beta-diversity were compared among sampling locations. Results Microbiota composition differed across sampling locations, with physiologically and anatomically distinct locations showing different relative abundances of 1137 observed sequence variants (SVs). An analysis of similarities showed that the lung was more similar to the nasopharynx (R-statistic = 0.091) than it was to the oropharynx (R-statistic = 0.709) or any other URT sampling location. Five distinct metacommunities were identified across all samples after clustering at the genus level using Dirichlet multinomial mixtures. This included a metacommunity found primarily in the lung and nasopharynx that was dominated by Mycoplasma. Further clustering at the SV level showed a shared metacommunity between the lung and nasopharynx that was dominated by Mycoplasma dispar. Other metacommunities found in the nostrils, tonsils, and oral microbiotas were dominated by Moraxella, Fusobacterium, and Streptococcus, respectively. Conclusions The nasopharyngeal bacterial microbiota is most similar to the lung bacterial microbiota in healthy cattle and therefore may serve as the primary source of bacteria to the lung. This finding indicates that the nasopharynx is likely the most important location that should be targeted when doing bovine respiratory microbiota research.
    A rapid, low cost, and highly sensitive SARS-CoV-2 diagnostic based on whole genome sequencing
    Brian Glenn St Hilaire, BioRxiv - 2020
    Abstract
    Early detection of infection with SARS-CoV-2 is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset1,2. Here, we introduce a low-cost, high-throughput method for diagnosis of SARS-CoV-2 infection, dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), that enhances sensitivity by aiming to amplify the entire SARS-CoV-2 genome rather than targeting particular viral loci, as in typical RT-PCR assays. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. We are able to reliably (>95% accuracy) detect SARS-CoV-2 at concentrations of 84 genome equivalents per milliliter, better than the reported limits of detection of almost all diagnostic methods currently approved by the US Food and Drug Administration. At higher concentrations, we are able to reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy. Such genome assemblies enable the spread of the disease to be analyzed much more effectively than would be possible with an ordinary yes/no diagnostic, and can help identify vaccine and drug targets. Using POLAR, a single person can process 192 samples over the course of an 8-hour experiment, at a cost of ~$30/patient, enabling a 24-hour turnaround with sequencing and data analysis time included. Further testing and refinement will likely enable greater enhancements in the sensitivity of the above approach.
    Influence of introduced arbuscular mycorrhizal fungiand phosphorus sources on plant traits, soil properties,and rhizosphere microbial communities in organiclegume-flax rotation
    Yunliang Li, Plant Soil - 2019
    Abstract
    Aims We identify P management strategies combiningarbuscular mycorrhizal fungal (AMF) inoculation withrock phosphate or composted manure for intensive or-ganic grain-production systems.Methods We measured the response of plants traits andsoil properties to the factorial combination of three ratesof organic-approved P sources applied in rotation phase-1of legume–flax cropping systems, and of granular AMFinoculant applied in the first, second, or both rotationphases, or not applied. Treatment combinations effectson the rhizosphere communities of AMF, fungi, andbacteria were tested by amplicon sequencing, in twopedoclimates.Results Inoculation had limited effects in both environ-ments. Composted manure decreased lentil yield, butincreased lentil N and P concentrations and soil P fertilityon the Chernozem, while increasing pea productivity onthe Luvisol. Composted manure applied in rotationphase-1 had a residual effect on flax productivity, N andP concentrations, and soil P fertility in both environ-ments. Rock phosphate reduced soil P fertility and flaxproductivity on the Gray Luvisol. The βdiversity of therhizosphere communities was unaffected by treatments,while the αdiversity of bacteria and AMF was altered byAMF inoculation and fertilization only in the GrayLuvisol. Correlations between microbial species andplant traits or soil properties were inconsistent, reflectingthe complex relationships among microbial community,plant identity, and environmental conditions.Conclusion Here, composted manure was more influ-ential than AMF inoculation and rock phosphate. Giventhe influence of environmental conditions, small fieldtrials are recommended before wide-scale adoption oftheir use.
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