Optimizing the extraction of human microbiome samples

The influence of the human microbiome on health is an important area of ongoing investigation for medicine and diagnostics. For these studies, it is crucial to have a reliable, reproducible extraction method which is able to extract DNA of all microorganisms with comparable efficiency, while simultaneously depleting inhibitory substances which might disturb subsequent downstream analysis. We present the results of DNA extraction and microbiome analysis from stool, saliva, and a variety of swabs (buccal, vaginal, rectal, skin) using the DNeasy PowerSoil Pro/QIAamp PowerFecal Pro kits, including automation options and process handling improvements. Materials/methods: Human microbiome samples (stool, saliva, and buccal, vaginal, rectal and skin swabs) were homogenized and microbial cells (including gram-negative and gram-positive bacteria, fungi, and archaea) were rapidly and efficiently lysed by bead beating. Inhibitory substances were removed through the use of Inhibitor Removal Technology (IRT). Thereafter, inhibitor-free DNA was purified and used for 16S rDNA sequencing experiments. Within these experiments, the effect of using inoculating loops for stool sampling was investigated, as well as the impact of automating the procedure. Results: The DNeasy PowerSoil Pro/QIAamp PowerFecal Pro Kits were able to efficiently extract microbial DNA with up to 20-fold higher yields compared to other methods.  The extracted samples showed no inhibition in PCR. The use of inoculation loops for stool sampling was shown to be feasible and accurate. 16S rDNA sequencing revealed distinct and complex communities within the different sample types. Conclusion: The DNeasy PowerSoil Pro and QIAamp PowerFecal Pro kits are an optimal method for extracting microbial DNA from human samples for microbiome analysis and can be used for a wide variety of sample types in addition to stool.

Learning Objectives:

1. Understand the challenges of extracting DNA from human samples for microbiome analysis; learn about the best technologies for accomplishing this

2. Learn about process optimization for DNA extraction

3. Learn about applications of the standard stool kits for the processing of different human sample types