Extended Data Fig. 3: Alpha-diversity indices of stool and stool-derived microbial communities.
Stool samples from five healthy donors (MB001–MB003, MB005 and MB006) were collected and immediately placed in an anaerobic chamber. Samples were mixed with 40% glycerol in PBS + 0.5 g of cysteine/liter, divided into aliquots in ~700-µl glycerol stocks and frozen at −80 °C (non-processed stool). Before freezing, we performed a 1:1,000 dilution of one aliquot per donor in 50 ml of mGAM medium and grew it anaerobically at 37 °C for 24 h (culture from fresh stool (24h)). We then mixed 800 µl of the culture with 200 µl of 50% (vol/vol) glycerol with palladium black (glycerol stock); we also diluted another 5 µl of the culture in 5 ml of mGAM and cultured it anaerobically at 37 °C for 24 h (culture from fresh stool (48 h)). After 10–14 weeks, we inoculated the glycerol stocks in 5 ml of mGAM, cultured them anaerobically at 37 °C for 24 h (culture from glycerol stock (24 h)), passaged them (1:1,000) in fresh mGAM and cultured them at 37 °C for another 24 h (culture from glycerol stock (48 h)). We harvested cell pellets from the cultures by centrifugation, extracted DNA and performed 16S rRNA amplicon sequencing. After read count rarefaction and exclusion of samples with low sequencing depth, we calculated the species richness and Shannon index of the samples. Compared to fresh stool samples, the alpha diversity in the stool-derived communities was reduced. However, the number of passages and cryopreservation did not seem to have a major effect on the diversity of the communities. Similar pilot tests should be carried out for each community. It is also recommended to check the long-term stability of the cryopreserved communities. Each experiment should include an unperturbed (solvent) control from the same cryotube. Stool material collection was approved by the EMBL Bioethics Internal Advisory Committee, and informed consent was obtained from all donors (BIAC2015-009).
