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

Microbiome

Takamasa Miura¹  Mamiko Shimamura¹  Yoshifumi Ohyama¹  Hiroko Kawasaki¹  Yoshihito Uchino¹  Atsushi Yamazoe¹  Dieter M. Tourlousse²  Masami Matsuda²  Yuji Sekiguchi²  Akiko Ohashi²  Keita Shiina²  Daisuke Miura²  Yoshiki Tanaka³  Takuya Fuchikami⁴  Hitomi Aoki⁴  Satoshi Kira⁴  Ken Kasahara⁵  Koji Narita⁵  Takeshi Naito⁶  Morie Nishiwaki⁶  Kazuyuki Fujii⁷  Keishi Kameyama⁸  Hirotaka Kumagai⁹  Mikiko Konda⁹  Jiro Kataoka^1,10  Masatomo Rokushima^1,11  Shingo Arioka^1,11  Miho Kuroiwa^1,11  Ryo Koyanagi^1,12  Jun Terauchi^1,13  Takayoshi Hisada^1,14  Shunsuke Takahashi^1,14  Mitsue Nishiyama^1,15  Moriya Ohkuma^1,16  Mitsuo Sakamoto^1,16 

[1] Biological Resource Center, National Institute of Technology and Evaluation (NITE), 292-0818, Kisarazu, Chiba, Japan;Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 305-8566, Tsukuba, Ibaraki, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Biofermin Pharmaceutical Co., Ltd., 650-0021, Kobe, Hyogo, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;CDM Center Division 4, Takara Bio Inc., 525-0058, Kusatsu, Shiga, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Chitose Laboratory Corp., 216-0041, Kawasaki, Kanagawa, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;H.U. Group Research Institute G.K., 192-0031, Hachioji, Tokyo, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Infectious Diseases Unit, Department of Medical Innovations, New Drug Research Division, Otsuka Pharmaceutical Co., Ltd., 771-0192, Tokushima, Tokushima, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Institute of Food Sciences and Technologies, Ajinomoto Co., Inc., 210-8681, Kawasaki, Kanagawa, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;JSR-Keio University Medical and Chemical Innovation Center, 160-8582, Shinjuku, Tokyo, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Japan Tobacco Inc., Minato, 105-6927, Tokyo, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Laboratory for Innovative Therapy Research, Shionogi and Co., Ltd., 561-0825, Toyonaka, Osaka, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Molecular Genetic Research Department, Advanced Technology Center, LSI Medience Corporation, 101-8517, Chiyoda, Tokyo, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Ono Pharmaceutical Co., Ltd., 541-8564, Osaka, Osaka, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;TechnoSuruga Laboratory Co., Ltd., 424-0065, Shizuoka, Shizuoka, Japan;Japan Microbiome Consortium (JMBC), 530-0011, Osaka, Osaka, Japan;Tsumura Kampo Research Laboratories, Tsumura & Co., 300-1192, Ami, Ibaraki, Japan;Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Research Center, 305-0074, Tsukuba, Ibaraki, Japan;

关键词: Human microbiome;    Metagenomics;    Gut microbiota;    Standardization;    Accuracy, reproducibility, and comparability;    Industrialization;    DNA extraction;    Library construction;   

DOI  :  10.1186/s40168-021-01048-3

来源: Springer

【 摘 要 】

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 Abstract

【 授权许可】

CC BY