Integrity of fecal microbiome biospecimen diversity and composition across various temperatures and time points
Abstract – The intestinal microbiome continues to be a growing area of research with benefits for human health. Studies involving collection of fecal specimens from human subjects usually employ collection protocols aimed at preserving microbial composition during storage of samples and subsequent DNA sequencing. These protocols dictate parameters such as the allowable time between collection and freezing, so as to minimize chances of alteration of the microbial populations and degradation of bacterial DNA. However, these guidelines vary greatly, and currently there is little consensus as to how long samples can truly be stored. In this study I sought to concretely examine the degradation of samples by evaluating the integrity of the gut microbiome in fecal specimens at different temperatures (-80°C, -20°C, 4°C, room temperature) across several time points (0, 3, 8, 11 days) after collection. Specimens were analyzed by 16S rRNA gene sequencing and curated using the DADA2 pipeline. For each sample, alpha diversity and microbial composition were assessed and compared. A total of 11,062,741 16S sequences across 26 samples were obtained. Linear discriminant analysis effect size was used to assess the effects of storage time and temperature on degradative changes to the sample. Notably, I found that bacterial diversity and composition remained relatively the same across all conditions. I conclude that the parameters for human fecal sample collection could potentially be relaxed in terms of allowable storage time and temperature. This may help to improve the practical feasibility of microbiome studies involving sample collection from human subjects.