A Comparison of Six DNA Extraction Protocols for 16S, ITS and Shotgun Metagenomic Sequencing of Microbial Communities
Year: 2022
Type: article
Abstract: 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 fungal ITS data, we compared five candidate DNA extraction kits against our standardized protocol for describing bacteria and archaea using 16S rRNA gene amplicon- and shotgun metagenomics sequencing. We present results 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, we found the MagMAX Microbiome kit 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. 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_kit_testing). Methods summary To allow for downstream applications involving fungi in addition to bacteria and archaea, we compared five DNA extraction kits with our previously established, standardized protocol for extracting DNA for microbial community analysis. Across ten diverse sample types, we found one extraction kit to perform comparably or better than our standardized protocol. Our conclusion is based on per-sample comparisons of DNA yield, the number of quality-filtered sequences generated, the limit of detection of microbial cells, microbial community alpha-diversity, beta-diversity, and taxonomic composition, and extent of well-to-well contamination.
Source: BioTechniques
Language: English
Topic: Gut microbiota and health
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,415
Funders: Semiconductor Research Corporation, Crohn's and Colitis Foundation of America, Crohn's and Colitis Foundation, Emerald Foundation, National Institutes of Health, National Institute of Food and Agriculture, National Institute on Aging, National Heart, Lung, and Blood Institute, Office of Naval Research, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, National Center for Complementary and Integrative Health
Awards: G2574213254, Mapping host-microbe-metabolite interactions in 3D to find diet-derived enhancers of immunity, G3462717068, G3760032604, Gut Liver Brain Biochemical Axis in Alzheimer's Disease, Alzheimer's Gut Microbiome Project, Integrated analysis of CVD risk in HIV: gut microbiota, immune function and metabolites, G6053108557, G6196693915, Translational Studies of Age-Associated Arterial Dysfunction, Western Diet and Aerobic Exercise: Role of the Gut Microbiome, Systems Biology Approach to Redefine Susceptibility Testing and Treatment of MDR Pathogens in the Context of Host Immunity, The Subgingival Microbiome and Impaired Glucose Regulation, San Diego IRACDA Scholars Program
OpenAlex ID: W4283065918