Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods.
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ABSTRACT:
SUBMITTER: Yao Y
PROVIDER: S-EPMC8085529 | biostudies-literature |
REPOSITORIES: biostudies-literature
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2020-05-19 | PXD015625 | Pride
Project description:The need to develop valid methods for sampling and analyzing fecal specimens for microbiome studies is increasingly important, especially for large population studies.Some of the most important attributes of any sampling method are reproducibility, stability, and accuracy. We compared seven fecal sampling methods [no additive, RNAlater, 70% ethanol, EDTA, dry swab, and pre/post development fecal occult blood test (FOBT)] using 16S rRNA microbiome profiling in two laboratories. We evaluated nine commonly used microbiome metrics: abundance of three phyla, two alpha-diversities, and four beta-diversities. We determined the technical reproducibility, stability at ambient temperature, and accuracy.Although microbiome profiles showed systematic biases according to sample method and time at ambient temperature, the highest source of variation was between individuals. All collection methods showed high reproducibility. FOBT and RNAlater resulted in the highest stability without freezing for 4 days. In comparison with no-additive samples, swab, FOBT, and 70% ethanol exhibited the greatest accuracy when immediately frozen.Overall, optimal stability and reproducibility were achieved using FOBT, making this a reasonable sample collection method for 16S analysis.Having standardized method of collecting and storing stable fecal samples will allow future investigations into the role of gut microbiota in chronic disease etiology in large population studies.
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Project description:The impact of the DNA extraction methods on the gut bacterial and fungal microbiota community recovery
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Project description:The establishment of human gut microbiota commences initially in utero. Meconium-the first fecal material passed after birth-can be used to study fetal gut contents; however, processing meconium samples for microbiome studies presents significant technical challenges. Meconium hosts a low biomass microbiome, is tar-like in texture and contains high concentrations of PCR inhibitors. This study aimed to evaluate four different DNA extraction methods to elucidate the most effective method for bacterial DNA recovery and sequencing analysis from first-pass meconium. Samples from five infants were collected and processed using the following extraction kits: (1) Qiagen QIAamp DNA Stool Mini (QS); (2) Qiagen QIAamp DNA Microbiome (QM); (3) MoBio PowerSoil (PS); (4) MoBio MagAttract PowerMicrobiome (PM). Additionally, Kit PM was employed with a double inhibitor removal treatment (IRT) step (PM2). Bacterial DNA recovery was assessed by qPCR. Any PCR inhibition in samples was measured by spiking DNA eluates with 0.1 ng of pure Streptococcus agalactiae (GBS) DNA followed by qPCR quantitation. Kit PM yielded the highest average total DNA yield (79.3 ng per gram of meconium). Samples extracted with kit PS had the highest detectable levels of 16S rRNA gene by qPCR. The ability of each kit to overcome PCR inhibition varied, with qPCR on GBS-spiked DNA from kits QS, QM, PS, and PM recovering 87.1, 91.0, 88.8, and 37.9% GBS DNA, respectively. Double IRT improved the performance of kit PM, increasing GBS recovery to 56.5%. However, once DNA yield was normalized to the level recovered with the other kits 100% of GBS DNA was detected, suggesting that levels of PCR inhibitors are related to DNA yield from kit PM. Ion Torrent 16S rRNA gene sequencing revealed a high level of inter-kit variation in meconium microbiome structure. In particular, kit QM showed a bias toward extracting Firmicute DNA, while the other kits extracted primarily Proteobacterial DNA. Choice of extraction kit greatly impacts on the ability to extract and detect bacterial DNA in meconium and on the microbiome community structure generated from these samples.
| S-EPMC5826226 | biostudies-literature
Project description:BackgroundNumerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly.ResultsIn this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1-26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis.ConclusionsThe evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.
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Project description:Highlights • Extractions from soil, plants, and water were tested for pesticide C and N CSIA.• Pesticide recoveries strongly varied among compounds and matrices properties.• Tested extraction methods caused no effect on δ13C and δ15N of pesticides.• C and N pesticide CSIA can be applied in situ to agricultural water samples.• Pesticide CSIA for soil and sediment samples are limited to source areas. Compound-specific isotope analysis (CSIA) is a powerful approach to evaluate the transformation of organic pollutants in the environment. However, the application of CSIA to micropollutants, such as pesticides, remains limited because appropriate extraction methods are currently lacking. Such methods should address a wide range of pesticides and environmental matrices, while recovering sufficient mass for reliable CSIA without inducing stable isotope fractionation. Here, we present simple extraction methods for carbon and nitrogen CSIA for different environmental matrices and six commonly used herbicides, i.e., atrazine, terbutryn, acetochlor, alachlor, butachlor, and S-metolachlor, and three fungicides, i.e., dimethomorph, tebuconazole, and metalaxyl. We examined the potential of several extraction methods for four types of soils or sediments, three types of environmental waters and aerial and root plant samples for multielement (ME)-CSIA.• Pesticide extraction recoveries varied depending on the physical characteristics of the pesticides and matrix properties for environmental water (77 to 87%), soil and sediment (35 to 82%), and plant (40 to 59%) extraction.• The tested extraction methods did not significantly affect the carbon and nitrogen stable isotope signatures of pesticides (Δ(13C) <0.9‰ for Δ(15N) <1.0‰). Graphical abstract Image, graphical abstract
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| S-EPMC7936037 | biostudies-literature
Project description:This paper reviews the currently used pretreatment methods for microplastics (MPs) analysis in soil and freshwater sediments, primarily sample processing, pretreatment, and characterization methods for MPs analysis. In addition, analytical tools (e.g., lab instruments), MPs characteristics, and MPs quantity, are included in this review. Prior to pretreatment, soil and sediment samples are typically processed using sieving and drying methods, and a sample quantity of <50 g was mostly used for the pretreatment. Density separation was commonly performed before organic matter removal. Sodium chloride (NaCl) and zinc chloride (ZnCl2) were most often used for density separation, and hydrogen peroxide (H2O2) oxidation was most frequently used to remove organic matter. Although advantages of each pretreatment method have been investigated, it is still challenging to determine a universal pretreatment method due to sample variability (e.g., sample characteristics). Furthermore, it is highly required to establish standard pretreatment methods that can be used for various environmental matrices, including air, water, and wastes as well as soil and sediment.
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