A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities
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Abstract
One goal among 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 we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.
Accession numbers
Raw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub ( github.com/justinshaffer/Extraction_test_MagMAX ).
Methods summary
To allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.
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SciScore for 10.1101/2020.11.13.370387: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Human urine samples included samples from female and male individuals. Table 2: Resources
Software and Algorithms Sentences Resources For shallow shotgun metagenomics data, raw sequence files were demultiplexed using BaseSpace (Illumina, La Jolla, CA), quality-filtered using Atropos [34] and human read depleted by alignment to human reference genome GRCh38 using bowtie2 [35]. BaseSpacesuggested: (BaseSpace, RRID:SCR_011881)bowtie2suggested: (Bowtie 2, RRID:SCR_016368)Results from OddPub: Thank you for sharing your code and data.
Results from LimitationRecog…SciScore for 10.1101/2020.11.13.370387: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Human urine samples included samples from female and male individuals. Table 2: Resources
Software and Algorithms Sentences Resources For shallow shotgun metagenomics data, raw sequence files were demultiplexed using BaseSpace (Illumina, La Jolla, CA), quality-filtered using Atropos [34] and human read depleted by alignment to human reference genome GRCh38 using bowtie2 [35]. BaseSpacesuggested: (BaseSpace, RRID:SCR_011881)bowtie2suggested: (Bowtie 2, RRID:SCR_016368)Results from OddPub: Thank you for sharing your code and data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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