FIG 1.

Overview of the experimental setup and alpha diversity of microbiomes. (A) Two pig feces and two sewage samples were divided into two large aliquots, of which one was spiked with a synthetic mock community composed of eight microorganisms that included two eukaryotes (Propionibacterium freudenreichii, Bacteroides fragilis, Staphylococcus aureus, Fusobacterium nucleatum, Escherichia coli, Salmonella enterica serovar Typhimurium, Cryptosporidium parvum, and Saccharomyces cerevisiae), respectively. Each of these eight large aliquots was divided into small aliquots that were stored at four different temperatures (−80°C, −20°C, 5°C, and 22°C) for six different durations of time (0 h, 16 h, 64 h, 4 months, 8 months, and 12 months). DNA was isolated at the three different time points in triplicate. Additional aliquots for samples P1 and S1 underwent repeated freeze-thaw cycles following 40 h, 64 h, and 88 h of storage duration. For details, see Materials and Methods. All samples were sequenced on an Illumina HiSeq. (B) Alpha diversity was determined (richness [Chao1], evenness [Pielou’s evenness], and diversity [Simpson]). The indices were calculated from the count table aggregated at the genus level. For the results of the duplicate sets of samples that also harbored the spiked mock community, see Fig. S3 in the supplemental material.