DA BEI ZHOU TUTORIAL MANUAL

D6300), containing cells of 8 species of bacteria (each making up 12%) and 2 yeast strains (each contributing 2%), was purchased from Zymo Research (Fig. Data DescriptionДNA extraction protocols ( Supplementary Table S1) on 2 types of biological samples, including a 10-species MMC and human fecal samples from 6 healthy individuals (Fig. On the basis of extractions from human fecal samples, we found that a time- and cost-effective kit-based protocol, protocol 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. On the basis of extractions of the MMC, we established a positive correlation between the bead size and extraction efficiency of yeast DNA, providing information for the selection of appropriate DNA extraction protocols for fungal-related studies. In the present study, we assessed the DNA extraction performance of 6 protocols on a microbial mock community (MMC) comprising 8 bacterial and 2 yeast strains, and on fecal samples from 6 healthy human individuals, using protocol Q as a reference method.

Additionally, assessment of fungal DNA extraction performance in fecal samples, the often neglected important players in the overall gut microbiome, is still scarce. However, there is still room for improvement to establish less labor-intensive and more cost-effective alternative standardized protocols, especially for large-scale gut microbiome studies.

DA BEI ZHOU TUTORIAL MANUAL

By evaluation of DNA quantity and quality, community diversity, and extraction efficiency of gram-positive and gram-negative bacteria, this study has proposed protocol Q, a manual protocol based on a modified version of Qiagen's QIAampВ® DNA Stool Mini Kit, as a standard protocol for human fecal bacterial DNA extraction. A recent shotgun sequencingвЂ“based benchmark study has comprehensively investigated bacterial extraction performances of 21 fecal DNA extraction protocols, including widely used extraction kits and nonвЂ“kit-based protocols. In comparison, shotgun metagenomic sequencing is a more accurate tool for analyzing the microbiota. However, considering the known effects of PCR conditions on amplification biases such as primers, specific hypervariable regions, and annealing temperature, amplicon sequencing is insufficient for accurately evaluating the quantitative performance of bacterial DNA extraction protocols. However, many studies have shown how different experimental processing pipelines affect the results and how especially DNA extraction affects the quantitative characterization of bacterial components, emphasizing the need for a standardized and robust protocol for profiling of the gut microbiota to enable true comparison between studies.Дuring 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. The composition of the gut bacterial community has been demonstrated to exhibit associations with multiple human diseases, including type 2 diabetes, obesity, and colorectal cancer.

The adult human gut harbors highly complex and diverse microbial communities, including bacteria, archaea, fungi, viruses, and protozoa. 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. In human fecal samples, the MagPure bacterial extraction performed as well as the standardized protocol Q but was faster and more cost-effective. The MMC results demonstrated that bead size was a determining factor for fungal and bacterial DNA yields. Extracting DNA revealed high reproducibility within all 6 protocols, but microbial extraction efficiencies varied. All samples were manually extracted and subjected to shotgun metagenomics sequencing.

We compared 6 DNA extraction protocols, MagPure Fast Stool DNA KF Kit B, Macherey Nagelв„ў NucleoSpinв„ўВ®Soil kit, Zymo Research Quick-DNAв„ў 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.

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