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

Abstract

Background: Gut microbiota analysis can help understand many diseases some of which, like inflammatory bowel diseases, obesityanddiabetesarealreadyestablishedandtheothersaretobe determined.Usingthistestfordiagnosticpurposesandestablishing personalizedcareseemstobecomearoutineprocedurethatneeds standardizationprotocols.Theaimofthisstudywastoanalyzethe changesingutmicrobiotacommunitiesinsamplestakenfromsingle case at different times and the effect of storing the samples in -20◦C before processing them. Methods&Materials:Ontheday1andtheday8,twostoolsamples 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 2hours and part of it was kept in -20 until day 8. The second stool sample was processed together with the frozen sample in 2hours. DNA was obtained with fecal DNA extraction kit (QIAGENE). The microbiota was determined using Ion16SrRNAMetagenomicKitonIonTorrentplatform.Theextractionweredoneasmentionedaboveandmetagenomicstudieswere doneforallsamplesinasinglerunandchip.Thecomparisonofthe 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%and19%infrozenand8thdaysamplesrespectively.Proteobacteria ratios were high in frozen and 8th day samples: 31% and 36%. Proteobacteria formed 7% of bacterial population in the 1st dayfreshsample.Tenericutes,ActinobacteriaandVerrucomicrobia were below 1% in all samples.