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

Microbiome

Yoshifumi Ohyama¹  Yoshihito Uchino¹  Mamiko Shimamura¹  Takamasa Miura¹  Hiroko Kawasaki¹  Atsushi Yamazoe¹  Akiko Ohashi²  Masami Matsuda²  Daisuke Miura²  Keita Shiina²  Dieter M. Tourlousse²  Yuji Sekiguchi²  Jun Terauchi³  Keishi Kameyama³  Takeshi Naito³  Mikiko Konda³  Morie Nishiwaki³  Ryo Koyanagi³  Satoshi Kira³  Masatomo Rokushima³  Hirotaka Kumagai³  Shingo Arioka³  Hitomi Aoki³  Koji Narita³  Kazuyuki Fujii³  Yoshiki Tanaka³  Jiro Kataoka³  Ken Kasahara³  Takayoshi Hisada³  Mitsue Nishiyama³  Miho Kuroiwa³  Takuya Fuchikami³  Shunsuke Takahashi³  Mitsuo Sakamoto⁴  Moriya Ohkuma⁴ 

[1] Biological Resource Center, National Institute of Technology and Evaluation (NITE);Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST);Japan Microbiome Consortium (JMBC);Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Research Center;

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

DOI  :  10.1186/s40168-021-01048-3

来源: DOAJ

【 摘 要 】

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. Video Abstract

【 授权许可】

Unknown