Standardization of gut microbiota analysis: Variability in samples taken at different times from single case and the effect of the freezing the sample

      Background: Gut microbiota analysis can help understand many diseases some of which, like inflammatory bowel diseases, obesity and diabetes are already established and the others are to be determined. Using this test for diagnostic purposes and establishing personalized care seems to become a routine procedure that needs standardization protocols. The aim of this study was to analyze the changes in gut microbiota communities in samples taken from single case at different times and the effect of storing the samples in -20 °C before processing them.
      Methods & Materials: On the day 1 and the day 8, two stool samples were obtained from a single case, 40-year-old woman using insulin for the control of her Type 1 Diabetes Mellitus. The first sample were processed in 2 hours and part of it was kept in -20 until day 8. The second stool sample was processed together with the frozen sample in 2 hours. DNA was obtained with fecal DNA extraction kit (QIAGENE). The microbiota was determined using Ion 16S rRNA Metagenomic Kit on Ion Torrent platform. The extraction were done as mentioned above and metagenomic studies were done for all samples in a single run and chip. The comparison of the results was done using t-test with SPSS (IBM) software.
      Results: Six phyla were determined: Bacteriodetes, Firmicutes, Proteobacteria, Tenericutes, Actinobacteria and Verrucomicrobia. Bacteriodetes and Firmicutes formed the majority of the bacterial population studied. Bacteriodetes were 57% in the 1st day fresh sample and 28% in the frozen one. Bacteriodetes ratio in 8th day sample was 44%. Firmicutes formed 35% in 1st day fresh sample, 38% and 19% in frozen and 8th day samples respectively. Proteobacteria ratios were high in frozen and 8th day samples: 31% and 36%. Proteobacteria formed 7% of bacterial population in the 1st day fresh sample. Tenericutes, Actinobacteria and Verrucomicrobia were below 1% in all samples.
      Conclusion: Gut microbiota analysis results although being variable, showed a basic level consistency. Microbiota testing can be a part of routine work up when standard protocols for sample preparation and processing are established.