Comparison between microbiome of “paleofeces” and soil matrix to establish endogeneity

The human microbiome is an incredibly diverse and highly variable pseudo-organ responsible for vitamin synthesis, nutrient processing, and immune development, making microbial commensals essential for human health. Modern gut microbiome studies are hindered by a myriad of cultural and environmental factors affecting their composition, including agricultural practices, medication, dietary preference, and food processing techniques that have influenced every population on the planet. Analysis of paleofecal deposits recovered from archaeological sites provide temporal insight into the evolutionary history of the gut microbiome unimpeded by modern cultural practices. Research utilizing paleofeces elucidates modern cultural practices contribution to the rising frequency of many human health conditions (metabolic disorders, autoimmune disease, autism, and mental health) connected to the gut microbiome. Additionally, analysis of ancient human microbiomes allows for identification of species undiscovered through modern microbial analyses which could aide in the resolution of phylogenetic relationships and help to identify key genes with functional benefits. While studies utilizing coprolites to discern the microbiome of ancient populations already exist, to date the validity of coprolite use in these studies has not been explicitly addressed. A process known as DNA leaching, in which DNA from decomposing remains seeps into the surrounding soil matrix, challenges the assumption that the microbial data recovered from coprolites is endogenous. The design of this project addresses the aforementioned assumption by recreating the process that occurs to paleofecal deposits using modern fecal samples. The modern samples permit an accurate assessment of the degree of contamination that occurs in paleofeces because a portion is collected immediately thereby preserving a snapshot of the microbiome of the individual when the deposit was made. It also provides an opportunity to examine the percentage of genomic data lost through the immediate desiccation process. Through the combination of these strategies, I will discern (on a preliminary level) whether the microbial data of paleofeces is endogenous or a result of soil microbial contamination that has occurred through DNA leaching. Volunteers deposited fecal samples into an isolated rock shelter in the US Southwest. Upon deposition, a portion of the sample was collected immediately using sterilized fecal collection tubes. The remainder of the sample was left to desiccate within the rock shelter for six months. The samples were then recovered along with samples taken from the surrounding soil matrix. Each sample was extracted with four pseudo replicates and prepared for sequencing of the 16s ribosomal gene to identify the microbial composition. The composition of each sample will be compared with the corresponding samples and analyzed using Bray-Curtis dissimilarity based methods to address the following questions: a) Is the composition of the desiccated fecal samples distinct from that of the microbiome of the surrounding soil samples? b) Is the microbial composition of the desiccated samples consistent with the fresh samples? c) How much microbial signature is lost between the fresh samples and the desiccated samples? d) How much contamination from the soil matrix is present in the desiccated fecal samples