Abstract
BackgroundValidation 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.ResultsIn 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.ConclusionsThe 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.14cWN4qpGoEHsF7NJvL5FBVideo
Highlights
Our increased knowledge and mechanistic understanding of the role of human microbiota in health and disease [1,2,3] has created numerous opportunities for developing new strategies to improve human health through beneficial modulation of its microbiome [4, 5]
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
Summary
Our increased knowledge and mechanistic understanding of the role of human microbiota in health and disease [1,2,3] has created numerous opportunities for developing new strategies to improve human health through beneficial modulation of its microbiome [4, 5]. Global interest in the industrialization and commercialization of the therapeutic potential of the human microbiome has surged in sectors related to diagnostics, drug development, food, personnel care products, etc. The Japan Microbiome Consortium (JMBC) was established by the industry in Japan to identify and address precompetitive needs to support and accelerate the development of commercially viable products for the microbiome market. To improve consistency and enhance confidence in the accuracy of measurement results, standardization of metagenomic analysis methods has been recognized as a pressing need by industrial and regulatory sectors [14]. Validation and standardization of methodologies for microbial community measurements by highthroughput 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
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