Abstract
BackgroundShotgun 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.ResultsWe 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 reported gut microbial differences between healthy adults.ConclusionsWe emphasize the importance of bead size selection for bacterial and fungal DNA extraction. More importantly, the performance of the novel protocol MP matched that of the recommended standardized protocol Q but consumed less time, was more cost-effective, and is recommended for further large-scale human gut metagenomic studies.
Highlights
Shotgun metagenomic sequencing has improved our understanding of the human gut microbiota
On the basis of extractions from human fecal samples, we found that a time- and cost-effective kit-based protocol, protocol MagPure Fast Stool DNA KF Kit B (MP), exhibited bacterial DNA extraction performance similar to protocol Q regarding DNA yield, bacterial community diversity, and relative abundances of gram-positive and gram-negative bacteria
By plotting the abundance distributions of selected abundant gut species, including 6 gram-positive species (Bifidobacterium adolescentis, Bifidobacterium longum, Faecalibacterium prausnitzii, Collinsella intestinalis, Streptococcus anginosus, and Streptococcus cristatus) and 6 gram-negative species (Alistipes putredinis, Bacteroides coprocola, Bacteroides dorei, Bacteroides dorei/vulgatus, Bacteroides ovatus, and Prevotella copri), we found that species-related quantitative biases between MOBIO DNeasy PowerSoil kit (PS) and the other protocols were consistent among all individuals (Supplementary Fig. S6)
Summary
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 These recommendations can be further refined by considering the time and cost demands in dealing with samples from very large human cohorts. During the past 2 decades, PCR-based amplicon sequencing, a flexible and cost-effective method to determine microbial composition, has greatly improved our understanding of the human microbiome. By evaluation of DNA quantity and quality, community diversity, and extraction efficiency of grampositive and gram-negative bacteria, this study has proposed protocol Q, a manual protocol based on a modified version of Qiagen’s QIAamp R DNA Stool Mini Kit, as a standard protocol for human fecal bacterial DNA extraction [11]. Assessment of fungal DNA extraction performance in fecal samples, the often neglected important players in the overall gut microbiome [16,17,18], is still scarce
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