Assessment of fecal DNA extraction protocols for metagenomic studies

作者：Fangming Yang, Jihua Sun, Huainian Luo, Huahui Ren, Hongcheng Zhou, Yuxiang Lin, Mo Han, Bing Chen, Hailong Liao, Susanne Brix, Junhua Li, Huanming Yang, Karsten Kristiansen, Huanzi Zhong · 发表于：GigaScience · 年份：2020 · DOI：10.1093/gigascience/giaa071 · 被引用次数：95 · 研究领域：Gut microbiota and health、Clostridium difficile and Clostridium perfringens research、Fecal contamination and water quality

BACKGROUND: Shotgun metagenomic sequencing has improved our understanding of the human gut microbiota. Various DNA extraction methods have been compared to find protocols that robustly and most accurately reflect the original microbial community structures. However, these recommendations can be further refined by considering the time and cost demands in dealing with samples from very large human cohorts. Additionally, fungal DNA extraction performance has so far been little investigated. RESULTS: We compared 6 DNA extraction protocols, MagPure Fast Stool DNA KF Kit B, Macherey Nagel™ NucleoSpin™®Soil kit, Zymo Research Quick-DNA™ Fecal/Soil Microbe kit, MOBIO DNeasy PowerSoil kit, the manual non-commercial protocol MetaHIT, and the recently published protocol Q using 1 microbial mock community (MMC) (containing 8 bacterial and 2 fungal strains) and fecal samples. All samples were manually extracted and subjected to shotgun metagenomics sequencing. Extracting DNA revealed high reproducibility within all 6 protocols, but microbial extraction efficiencies varied. The MMC results demonstrated that bead size was a determining factor for fungal and bacterial DNA yields. In human fecal samples, the MagPure bacterial extraction performed as well as the standardized protocol Q but was faster and more cost-effective. Extraction using the PowerSoil protocol resulted in a significantly higher ratio of gram-negative to gram-positive bacteria than other protocols, which might contribute to...