Moreover,

since other next generation sequencing platforms will allow a greater sequencing depth, this may allow a deeper characterization of the microbial community and could reveal additional differences in the microbial community composition for the various conditions measured in this study. Finally, our study also reveals that microbial disruption by bead-beating allows greater detection of Gram-positive bacteria such as Blautia (Firmicutes phylum) and Bifidobacterium (Actinobacteria phylum), commonly detected in human PI3K inhibitor stools. In conclusion, the hydration of faecal samples and their degree of homogenisation do not significantly alter their microbial community composition and structure. However, although the mechanical disruption of microbial cells causes genomic DNA degradation in simulated diarrhoeic stool samples, our findings confirm that this step is necessary for the detection of Gram-positive bacteria such as Blautia and Bifidobacterium. Methods Ethics statement Subjects provided their written consent to participate see more in this study, and the Institutional Review Board of the Vall d’Hebron Hospital (Barcelona, Spain)

approved this consent procedure. Sample collection protocol Stools were collected from eight healthy participants. The collection protocol involved providing participants with an ice bag containing an emesis basin (Ref. 104AA200, PRIM S.A, Spain), a 50-mL sterile sampling bottle (Ref. 409526.1, Deltalab, Spain), a sterile spatula (Ref. 441142.2, Deltalab, Spain), and gloves (Additional file very 3: Figure S2) during their visit to the laboratory. For the purpose of stool collection, the participants were instructed to do the following once at home: 1) use the emesis basin to collect the stool; 2) after the deposit, transfer it to the sampling bottle ensuring no homogenisation; 3) take it to the lab within the first 3 hours after deposit; and 4) in the laboratory, the samples were processed as mentioned in the experimental design, and then the samples were stored at -80°C. Naming convention Since

the samples from same individuals were used to test different factors that could affect microbial composition, a labeling nomenclature had to be settled down as indicated in Table 1. The “D” stands for “diarrhoea” in the water content study. The “L” stands for “layer”, “O” for “outer”" and “I” for the “inner”" layer of the stool, and “H” for “homogenised stool” in the homogenisation evaluation. The “P” stands for samples that contained PBS to simulate diarrhoea not undergoing bead-beating, while “B” stands for samples that did not contain PBS, but underwent bead-beating. Samples with the “C” label are controls that did not contain PBS and did not undergo bead-beating. The numbers 1–8 signify the 8 different volunteers. Genomic DNA extraction To evaluate the need for stool homogenisation during collection, aliquots (250 mg) of each sample were suspended in 0.1 M Tris (pH 7.