Methods for Improving Human Gut Microbiome Data by Reducing Variability through Sample Processing and Storage of Stool

Abstract

Gut microbiome community analysis is used to understand many diseases like inflammatory bowel disease, obesity, and diabetes. Sampling methods are an important consideration for human microbiome research, yet are not emphasized in many studies. In this study, we demonstrate that the preparation, handling, and storage of human faeces are critical processes that alter the outcomes of downstream DNA-based bacterial community analyses via qPCR. We found that stool subsampling resulted in large variability of gut microbiome data due to different microenvironments harbouring various taxa within an individual stool. However, we reduced intra-sample variability by homogenizing the entire stool sample in liquid nitrogen and subsampling from the resulting crushed powder prior to DNA extraction. We experimentally determined that the bacterial taxa varied with room temperature storage beyond 15 minutes and beyond three days storage in a domestic frost-free freezer. While freeze thawing only had an effect on bacterial taxa abundance beyond four cycles, the use of samples stored in RNAlater should be avoided as overall DNA yields were reduced as well as the detection of bacterial taxa. Overall we provide solutions for processing and storing human stool samples that reduce variability of microbiome data. We recommend that stool is frozen within 15 minutes of being defecated, stored in a domestic frost-free freezer for less than three days, and homogenized prior to DNA extraction. Adoption of these simple protocols will have a significant and positive impact on future human microbiome research.

Fig 1. Bacterial taxa abundance differ in the inside compared to the outside microenvironments of stool.

A single stool was subsampled five times from the inside environment and five times from the outer environment, DNA was extracted and used to compare bacterial taxa via qPCR. Firmicutes and Bifidobacteria spp. were decreased in the outside microenvironment compared to the inside microenvironment of the stool. *, p = 0.03.

Fig 2. The mean variances of bacterial taxa are lower in homogenized subsamples compared to non-homogenized stool subsamples.

The variance values were calculated for each of the bacterial taxa tested using qPCR from five subsamples where the stool was homogenized by crushing on liquid nitrogen into a fine powder and compared to stool not homogenized. The mean variance was calculated by taking the average of the variances determined for each bacteria taxa from the four subjects that were examined.

Fig 3. Stool storage at room temperature alters the abundance of bacterial taxa.

Ten subsamples from the same stool were either stored at room temperature for 15 minutes or for 30 minutes, followed by DNA extraction and used to compare bacterial taxa via qPCR. Bacteroidetes detection decreased after 30 minutes at room temperature, whereas Firmicutes increased after 30 minutes. *, p > 0.05.

Fig 4. Stool storage in a domestic frost-free freezer affects the abundance of bacterial taxa.

A homogenized stool sample was stored in a domestic freezer for 0, 3,7,14, and 30 days, DNA was extracted and used for qPCR to compare bacterial taxa abundance. All bacterial taxa tested showed some change in abundance by day 30. *, p < 0.05.

Fig 5. Freeze-thawing stool up to four times does not affect bacterial taxa abundance.

A homogenized stool sample was subject to a series of up to five consecutive full freeze-thaw cycles, DNA was extracted and used for qPCR to compare bacteria taxa abundance. There were no changes of bacterial taxa abundance until the 5^th freeze thaw cycle where Bacteroidetes were increased and Enterobactericeae decreased. *, p < 0.05.

Fig 6. Stool stored in nucleic acid stabilizer prior to processing did not protect against bacterial taxa changes.

Stool was either stored with or without RNAlater (Qiagen) prior to freezing and then processing stool samples. Detection of Firmicutes, Lactobacillus spp. and Bifidobacteria spp. was reduced after storage in RNAlater. *, p = 0.05.