A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities.
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ABSTRACT:
SUBMITTER: Shaffer JP
PROVIDER: S-EPMC7931620 | biostudies-literature | 2021 Jan
REPOSITORIES: biostudies-literature
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A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities.
Shaffer Justin P JP Marotz Clarisse C Belda-Ferre Pedro P Martino Cameron C Wandro Stephen S Estaki Mehrbod M Salido Rodolfo A RA Carpenter Carolina S CS Zaramela Livia S LS Minich Jeremiah J JJ Bryant MacKenzie M Sanders Karenina K Fraraccio Serena S Ackermann Gail G Humphrey Gregory G Swafford Austin D AD Miller-Montgomery Sandrine S Knight Rob R
BioTechniques 20210129 3
One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols ...[more]
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Project description:Microbial communities contain a broad phylogenetic diversity of organisms; however, the majority of methods center on describing bacteria and archaea. Fungi are important symbionts in many ecosystems and are potentially important members of the human microbiome, beyond those that can cause disease. To expand our analysis of microbial communities to include data from the fungal internal transcribed spacer (ITS) region, five candidate DNA extraction kits were compared against our standardized protocol for describing bacteria and archaea using 16S rRNA gene amplicon- and shotgun metagenomics sequencing. The results are presented considering a diverse panel of host-associated and environmental sample types and comparing the cost, processing time, well-to-well contamination, DNA yield, limit of detection and microbial community composition among protocols. Across all criteria, the MagMAX Microbiome kit was found to perform best. The PowerSoil Pro kit performed comparably but with increased cost per sample and overall processing time. The Zymo MagBead, NucleoMag Food and Norgen Stool kits were included.
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Project description:In the last five years, high-throughput metabolomics has significantly advanced scientific research and holds the potential to promote strides in the fields of clinical metabolomics and personalized medicine. While innovations in the field of flow-injection mass spectrometry and three-minute metabolomics methods now allow investigators to process hundreds to thousands of samples per day, time-sensitive clinical applications, particularly in the emergency department, are limited by a lack of rapid extraction methods.Here we characterized the efficacy of fast liquid-liquid extractions for characterization of hydrophilic compounds through ultra-high-pressure liquid chromatography/mass spectrometry. Internal stable-isotope-labeled standards were used to quantitatively characterize markers of energy and oxidative metabolism in human whole blood, plasma and red blood cells - three common matrices of clinical relevance.For all the tested matrices, vortexing time (4-60 min) did not significantly affect extraction yields for the tested hydrophilic metabolites. Coefficients of variations <<20% for all tested compounds, except for the redox-sensitive metabolite cystine (accumulating over time). Internal standards and second extractions confirmed recoveries >80% for all tested metabolites, except for basic amino acids and polyamines, which showed reproducible yields ranging from 50 to 75%. Global profiling and absolute quantitation of 24 metabolites revealed similarities between the plasma and red blood cell metabolomes.Rapid extraction (~4 min) of hydrophilic compounds is a viable and potentially automatable strategy to perform quantitative analysis of whole blood, plasma and red blood cells for research or clinical applications.
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