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Comparison of Swabbing Solution Volume and gDNA Extraction Kits on DNA Recovery from Rigid Surface.

ABSTRACT: For bacteria sampling studies, various collection methods have been used to identify bacteria. To obtain accurate information about bacteria, high quality samples should be obtained. In order to obtain a high quality sample, a stable and large number of DNA copies must be collected. This study compared the efficiency of different methods of bacterial gDNA extraction and bacteria collection according to swabbing solution volumes and types. The efficiency of bacterial genomic DNA extraction was compared using a AccuPrep^® Genomic DNA Extraction kit, a QIAamp^® DNA Mini kit, and a MOBIO^® DNeasy PowerSoil kit. The DNA Mini kit was shown to extract the highest amount of gDNA, and sub-experiments were conducted using this kit. Phosphate-buffered saline and phosphate-buffered saline with 0.1% Tween 20 were used as collection solutions of various volumes (0, 40, 50, 60, 70, 80, 90, 100, 110, and 120 μL) using cotton swabs. Bacteria collection efficiency was highest when 70 μL PBS was used. The target strains collected in this experiment were Staphylococcus aureus and Escherichia coli, and these were quantified using the number of colony-forming units, DNA concentrations, and the number of DNA copies. These results can be used to efficiently bacterial collection for experiments in various fields.

SUBMITTER: Ok YJ

PROVIDER: S-EPMC7105554 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Describing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 ?m in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar). FINDINGS: We evaluated 1 salt precipitation method (MasterPure™ Complete RNA Purification Kit, Epicentre), 2 Phenol based extraction methods (mirVana Kit, Ambion, and Trizol Plus Kit, Invitrogen), 1 paramagnetic bead extraction method (RNA Tissue kit, GeneMole) and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix (Phenol-free Total RNA Purification Kit, Amresco, and ZR MicroPrep Kit, Zymo Research). The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen). CONCLUSIONS: We present an optimized extraction protocol for single and small numbers of Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.

Project description:IntroductionAdvancements in DNA extraction and sequencing technologies have been fundamental in deciphering the significance of the microbiome related to human health and pathology. Whole metagenome shotgun sequencing (WMS) is gaining popularity in use compared to its predecessor (i.e., amplicon-based approaches). However, like amplicon-based approaches, WMS is subject to bias from DNA extraction methods that can compromise the integrity of sequencing and subsequent findings. The purpose of this study was to evaluate systematic differences among four commercially available DNA extraction kits frequently used for WMS analysis of the microbiome.MethodsOral, vaginal, and rectal swabs were collected in replicates of four by a healthcare provider from five participants and randomized to one of four DNA extraction kits. Two extraction blanks and three replicate mock community samples were also extracted using each extraction kit. WMS was completed with NovaSeq 6000 for all samples. Sequencing and microbial communities were analyzed using nonmetric multidimensional scaling and compositional bias analysis.ResultsExtraction kits differentially biased the percentage of reads attributed to microbial taxa across samples and body sites. The PowerSoil Pro kit performed best in approximating expected proportions of mock communities. While HostZERO was biased against gram-negative bacteria, the kit outperformed other kits in extracting fungal DNA. In clinical samples, HostZERO yielded a smaller fraction of reads assigned to Homo sapiens across sites and had a higher fraction of reads assigned to bacterial taxa compared to other kits. However, HostZERO appears to bias representation of microbial communities and demonstrated the most dispersion by site, particularly for vaginal and rectal samples.ConclusionsSystematic differences exist among four frequently referenced DNA extraction kits when used for WMS analysis of the human microbiome. Consideration of such differences in study design and data interpretation is imperative to safeguard the integrity of microbiome research and reproducibility of results.

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Comparison of Swabbing Solution Volume and gDNA Extraction Kits on DNA Recovery from Rigid Surface.

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