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

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

Cardiorespiratory fitness as a predictor of intestinal microbial diversity and distinct metagenomic functions

Background: Reduced microbial diversity in human intestines has been implicated in various conditions such as diabetes, colorectal cancer, and inflammatory bowel disease. The role of physical fitness in the context of human intestinal microbiota is currently not known. We used high-throughput sequencing to analyze fecal microbiota of 39 healthy participants with similar age, BMI, and diets but with varying cardiorespiratory fitness levels. Fecal short-chain fatty acids were analyzed using gas chromatography. Results We showed that peak oxygen uptake (VO2peak), the gold standard measure of cardiorespiratory fitness, can account for more than 20 % of the variation in taxonomic richness, after accounting for all other factors, including diet. While VO2peak did not explain variation in beta diversity, it did play a significant role in explaining variation in the microbiomes’ predicted metagenomic functions, aligning positively with genes related to bacterial chemotaxis, motility, and fatty acid biosynthesis. These predicted functions were supported by measured increases in production of fecal butyrate, a short-chain fatty acid associated with improved gut health, amongst physically fit participants. We also identified increased abundances of key butyrate-producing taxa (Clostridiales, Roseburia, Lachnospiraceae, and Erysipelotrichaceae) amongst these individuals, which likely contributed to the observed increases in butyrate levels. Conclusions Results from this study show that cardiorespiratory fitness is correlated with increased microbial diversity in healthy humans and that the associated changes are anchored around a set of functional cores rather than specific taxa. The microbial profiles of fit individuals favor the production of butyrate. As increased microbiota diversity and butyrate production is associated with overall host health, our findings warrant the use of exercise prescription as an adjuvant therapy in combating dysbiosis-associated diseases.Health and Social Development, Faculty of (Okanagan)Arts and Sciences, Irving K. Barber School of (Okanagan)Biology, Department of (Okanagan)Health and Exercise Sciences, School of (Okanagan)ReviewedFacult

Homogenized stool subsamples have less variance compared to non-homogenized stool subsamples.

<p>Mean, standard deviation and variance values for each of the bacterial taxa detected via qPCR collected from four different subjects`stool subsamples that had been homogenized on liquid nitrogen or not homogenized. Levene's p-values are reported where a significant p-value denotes whether variance is significantly different between groups; the p value is italicized if the non-homogenized subsamples (underlined) have significantly higher variance compared to the homogenized subsamples. The averaged mean, standard deviation and variance between the four subjects is included in the right column (bolded text; large variance seen in non-homogenized subsamples).</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

RNAlater reduces DNA yields from stool samples.

<p>DNA extracted from samples not stored in RNAlater was significantly greater (p<0.0001) than samples stored in RNAlater.</p

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

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

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

<p>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.</p

List of primers used in this study.

<p>List of primers used in this study.</p