Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements

Abstract

Background Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. Results In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. Conclusions The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products.

Habitual Dietary Intake Affects the Altered Pattern of Gut Microbiome by Acarbose in Patients with Type 2 Diabetes

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“…However, low biomass alone does not explain the strong taxon effect on read assignment. We suspect that the most important cause for the strong taxon effects is likely caused by differences in DNA yields among different taxa (Sato et al, 2019;Schiebelhut, Abboud, Daglio, Swift, & Dawson, 2017;Tourlousse et al, 2021).…”

Health Effects of Drinking Water Produced from Deep Sea Water: A Randomized Double-Blind Controlled Trial

“…Following this, DNA was extracted from the suspension using the Magtration System 12GC and GC series MagDEA DNA 200 (Precision System Science Co., Ltd., Matsudo, Japan). The final DNA concentration (10 ng/μL) was subjected to the analysis of the microbial community structure by terminal restriction fragment length polymorphism and next-generation sequencing using the MiSeq system (Illumina, San Diego, CA, USA) at TechnoSuruga Laboratory Co., Ltd. [ 29 , 30 , 32 ]. Bioinformatic analysis was performed using the Ribosomal Database Project (RDP) Multiclassifier tool and Metagenome@KIN software (World Fusion Co., Ltd., Tokyo, Japan) based on data from bacterial species as determined by RDP taxonomic analysis.…”