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Background  To initiate fecal and oral collections in prospective cohort studies for microbial analyses, it is essential to understand how field conditions and geographic differences may impact microbial communities. This study aimed to investigate the impact of fecal and oral sample collection methods and room temperature storage on collection samples for studies of the human microbiota.    Results  We collected fecal and oral samples from participants in two Iranian cohorts located in rural Yazd ( n  = 46) and urban Gonbad ( n  = 38) and investigated room temperature stability over 4 days of fecal (RNA later  and fecal occult blood test [FOBT] cards) and comparability of fecal and oral (OMNIgene ORAL kits and Scope mouthwash) collection methods. We calculated interclass correlation coefficients (ICCs) based on 3 alpha and 4 beta diversity metrics and the relative abundance of 3 phyla. After 4 days at room temperature, fecal stability ICCs and ICCs for Scope mouthwash were generally high for all microbial metrics. Similarly, the fecal comparability ICCs for RNA later  and FOBT cards were high, ranging from 0.63 (95% CI: 0.46, 0.75) for the relative abundance of  Firmicutes  to 0.93 (95% CI: 0.89, 0.96) for unweighted Unifrac. Comparability ICCs for OMNIgene ORAL and Scope mouthwash were lower than fecal ICCs, ranging from 0.55 (95% CI: 0.36, 0.70) for the Shannon index to 0.79 (95% CI: 0.69, 0.86) for Bray-Curtis. Overall, RNA later , FOBT cards and Scope mouthwash were stable up to 4 days at room temperature. Samples collected using FOBT cards were generally comparable to RNA later  while the OMNIgene ORAL were less similar to Scope mouthwash.    Conclusions  As microbiome measures for feces samples collected using RNA later , FOBT cards and oral samples collected using Scope mouthwash were stable over four days at room temperature, these would be most appropriate for microbial analyses in these populations. However, one collection method should be consistently since each method may induce some differences.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12866-021-02387-9.

Comparison of fecal and oral collection methods for studies of the human microbiota in two Iranian cohorts