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:BackgroundSince the emergence of next generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA extraction methods using whole blood sampled under varying conditions from 20 wild carnivores.ResultsDespite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 - 18.4 μg) with varying integrity (RIN 4.6 - 7.7), the latter being significantly affected by the storage and extraction method used. We observed a significant overall effect of the extraction method on DNA contamination. One particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if libraries were not depleted of hemoglobin prior to sequencing.ConclusionBy carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species.
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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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