Experimental Factors Influence Diversity Metrics of the Gut Microbiome in Laboratory Mice

Abstract
Introduction

Gut microbiome studies often overlook experimental factors that could influence gut microbiome diversity and could impact findings. Large-scale studies investigating these experimental factors are lacking. Thus, we aimed to determine which experimental factors influence the gut microbiome diversity in pre-clinical animal model studies.

Methods

We extracted DNA and sequenced the V4 region of the 16S rRNA gene of a total of 538 samples from various sections of the gastrointestinal tract of 303 young and aged male and female C57BL/6J mice of three different genotypes on five diets from three animal house facilities. As a proof-of-concept in a disease model, some mice were treated with sham or angiotensin II, a commonly studied agent used as a hypertension model. Some samples were sequenced twice as a matched-comparison group.

Results

Using over 17 million sequencing reads, we found that experimental factors such as animal house facility, genotype, diet, age, sex, sampling site, and technical factor (i.e., sequencing batch) affected both α- and β-diversity (weighted and unweighted UniFrac), and were associated with compositional changes in the microbiome at varying magnitude, with diet and sampling site having the largest effect. After adjustment by these factors, treatment with angiotensin II had no impact on α-diversity and was only significant in unweighted UniFrac (presence/absence of bacteria) analyses.

Conclusion

Our data identified several key experimental and technical factors that affect the gut microbiome in laboratory mice. Our findings support that not accounting or adjusting for these factors may lead to false-positive discoveries and non-biologically relevant findings in the gut microbiome field.