Human faecal collection methods demonstrate a bias in microbiome composition by cell wall structure.

Clinical trial faecal collections present challenges through geographical spread and inexperienced participants. Collection techniques have been developed and tested to overcome these challenges, but previous studies investigating these techniques have demonstrated a highly variable capacity for sample preservation. Furthermore, these studies typically only examine either preservation of genetic content or metabolites, not both. This study investigated the Stool Nucleic Acid Collection and Preservation Tube (Norgen BioTek Corp) for the preservation of both microbial DNA and microbial organic acid metabolites in human faecal samples when compared to frozen samples. Twenty six healthy adult participants were instructed to collect a bowel movement, subsample into collection tubes and immediately transfer the remaining bulk to -20 °C storage. Resulting organic acid concentrations remained comparable across methods when the preservation tubes were used correctly. The 16S rRNA gene sequencing data revealed twenty significantly different bacterial genera between the two collection methods. Ten Gram-negative genera were more abundant in the collection tubes, and ten Gram-positive genera were more abundant in the fresh frozen samples. This study has illustrated that faecal collection methods bias the microbial community profile according to Gram status and this should be considered when designing studies that collect and store human faecal samples.