Critical evaluation of preservation methods for metagenomic analysis of faecal microbiomes

Objective The ability to preserve microbial communities [DP1] in faecal samples is essential as increasing numbers of studies seek to use the gut microbiome to identify biomarkers of disease. Here we rigorously evaluate five room temperature (RT) preservation methods to accurately represent species and functional profiles from faecal metagenomes compared to the commonly accepted best practice of flash-freezing.Design The methods evaluated were OMNIgene-GUT, RNAlater, LifeGuard, BBL CultureSwab, and the Copan FLOQSwab in an active drying tube (FLOQSwab-ADT) for the maximum recommended storage time at RT, then at -20?C until four weeks[DP2] [AP3] [AP4] . Each [DP5] method was assessed using six replicate faecal samples from five participants, totalling 180 samples. We further evaluated 240 samples across ten participants with the FLOQSwab-ADT to determine community stability after storage at -20?C, RT, and 50?C for four weeks compared to fresh controls. All samples were sequenced using shotgun metagenomics and gut microbiome profiles were assessed for technical and compositional (species and functional) reproducibility. Results FLOQSwab-ADT had the best combined performance followed by RNAlater and OMNIgene-GUT. LifeGuard and the BBL swab had unpredictable outgrowth of Escherichia species in at least one replicate from each participant. Additionally, FLOQSwab-ADT accurately preserved species and functional profiles at all temperatures tested relative to fresh controls. Conclusions FLOQSwab-ADT is a simple and robust liquid-free sampling method well-suited for direct-to-consumer and research-focused faecal sample preservation. BBL swab and LifeGuard are not recommended for RT preservation of faecal samples.