Human stool preservation impacts taxonomic profiles in 16S rRNA gene-based metagenomics studies

Background: Microbiotas play critical roles in human health, yet in most cases scientists lack standardized and reproducible methods. To date, stool sample preservation remains a source of technological bias in metagenomic sequencing, despite newly developed storage solutions. Studying those biases will help in identifying the appropriate stool preservation approach to promote the implementation of standard operating procedure and improve comparability across multiple microbiome studies. Results: Here, we conducted a comparative study of 10 storage methods for human stool over a 15-day period of storage at fluctuating temperatures. We first compared the performance of each stabilizer with observed bacterial composition variation within the same specimen. Then, we identified the nature of the observed variations to determine which bacterial populations were more impacted by the stabilizer. We found that DNA stabilizers display various stabilizing efficacies and affect the recovered bacterial profiles. Furthermore, our results showed that the bias associated with the stabilizers can be linked to the phenotypical characteristics of the bacterial populations present in the studied samples. Conclusions: Although newly developed storage solutions have improved our capacity to stabilize stool microbial content over time, they are nevertheless not devoid of biases. We therefore recommend considering the nature of these technological biases before claiming whether some microbes are beneficial or even deleterious to human health.