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

• DOI: 10.1186/s40168-021-01048-3
• Publication Date: 2021-04-29T00:44:40Z
• Authors: Dieter M. Tourlousse; Koji Narita; Takamasa Miura; Mitsuo Sakamoto; Akiko Ohashi; Keita Shiina; Masami Matsuda; Daisuke Miura; Mamiko Shimamura; Yoshifumi Ohyama; Atsushi Yamazoe; Yoshihito Uchino; Keishi Kameyama; Shingo Arioka; Jiro Kataoka; Takayoshi Hisada; Kazuyuki Fujii; Shunsuke Takahashi; Miho Kuroiwa; Masatomo Rokushima; Mitsue Nishiyama; Yoshiki Tanaka; Takuya Fuchikami; Hitomi Aoki; Satoshi Kira; Ryo Koyanagi; Takeshi Naito; Morie Nishiwaki; Hirotaka Kumagai; Mikiko Konda; Ken Kasahara; Moriya Ohkuma; Hiroko Kawasaki; Yuji Sekiguchi; Jun Terauchi
• Keywords: 0301 basic medicine; 0303 health sciences; Microbiota; QR100-130; Methodology; Reproducibility of Results; Gut microbiota; DNA; Sequence Analysis, DNA; Industrialization; Reference Standards; Human microbiome; Standardization; Microbial ecology; 03 medical and health sciences; Humans; Metagenomics; Accuracy, reproducibility, and comparability

AbstractBackgroundValidation 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.