Supporting data for "Assessment of fecal DNA extraction protocols for metagenomic studies"

Shotgun metagenomic sequencing has greatly improved our understanding of the human gut microbiota. Efforts have been made to evaluate the performance of various DNA extraction methods to recommend protocols that robustly and most accurately reflect the original microbial community structures. However, so far recommended standardized bacterial DNA extraction protocols still may be improved, especially considering future demands in relation to time and cost dealing with samples from very large cohorts. Additionally, fungal DNA extraction performance has so far been little investigated. <br>Here, we compared six DNA extraction protocols, MagPure Fast Stool DNA KF Kit B (MP), Macherey Nagel™ NucleoSpin™® Soil kit (MN), Zymo Research Quick-DNA™ Fecal/Soil Microbe kit (ZYMO), MOBIO DNeasy PowerSoil kit (PS), the manual protocol MetaHIT, and the recently published protocol Q using one microbial mock community (MMC) (containing eight bacterial and two fungal strains) and fecal samples. Evaluation of results using the MMC demonstrated that bead size was a determining factor for fungal and bacterial DNA yields. Evaluation of human fecal samples revealed that the bacterial extraction performance of protocol MP matched that of the standardized protocol Q, but consumed less time and was more cost-effective. Extraction using the protocol PS resulted in a significantly higher ratio of gram-negative to gram-positive bacteria than other protocols, which might potentially contribute to reported gut microbial differences between healthy US, Chinese and Danish adults, where fecal DNA samples were extracted using protocols PS and MetaHIT. <br>We reveal the importance of bead size selection for bacterial and fungal DNA extraction. More importantly, we demonstrate that the novel, time- and cost-effective protocol MP in terms of consistency and performance was equal to standardized protocol Q, and we recommend the use of MP for further large-scale metagenomic studies.