1. Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements
- Author
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Ryo Koyanagi, Akiko Ohashi, Jun Terauchi, Shingo Arioka, Takamasa Miura, Daisuke Miura, Yoshiki Tanaka, Hitomi Aoki, Keita Shiina, Miho Kuroiwa, Mitsuo Sakamoto, Dieter M. Tourlousse, Shunsuke Takahashi, Keishi Kameyama, Atsushi Yamazoe, Moriya Ohkuma, Masami Matsuda, Masatomo Rokushima, Takuya Fuchikami, Mikiko Konda, Hirotaka Kumagai, Takayoshi Hisada, Takeshi Naito, Ken Kasahara, Hiroko Kawasaki, Jiro Kataoka, Koji Narita, Mitsue Nishiyama, Morie Nishiwaki, Yoshifumi Ohyama, Yuji Sekiguchi, Kazuyuki Fujii, Satoshi Kira, Mamiko Shimamura, and Yoshihito Uchino
- Subjects
Microbiology (medical) ,Library construction ,Standardization ,Transferability ,Context (language use) ,Gut microbiota ,Biology ,Industrialization ,Microbiology ,Human microbiome ,Microbial ecology ,03 medical and health sciences ,Humans ,Microbiome ,Accuracy, reproducibility, and comparability ,DNA extraction ,030304 developmental biology ,Profiling (computer programming) ,0303 health sciences ,030306 microbiology ,Microbiota ,QR100-130 ,Methodology ,Reproducibility of Results ,DNA ,Sequence Analysis, DNA ,Reference Standards ,Data science ,Metagenomics - 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.
- Published
- 2021