Claire Cartozzo

While many studies have developed microbial succession‐based models for the prediction of postmortem interval (PMI) in terrestrial systems, similar well‐replicated long‐term decomposition studies are lacking for aquatic systems. Therefore, this study sought to identify temporal changes in bacterial community structure associated with porcine skeletal remains (n = 198) for an extended period in a fresh water lake. Every ca. 250 ADD, one cage, containing 5 ribs and 5 scapulae, was removed from the lake for a total of nineteen collections. Water was also sampled at each interval. Variable region 4 (V4) of 16S rDNA was amplified and sequenced for all collected samples using Illumina MiSeq FGx Sequencing platform; resulting data were analyzed with the mothur (v1.39.5) and R (v3.6.0). Bacterial communities associated with ribs differed significantly from those associated with scapulae. This difference was mainly attributed to Clostridia, Holophagae, and Spirochaete relative abundances. For each bone type, α‐diversity increased with ADD; similarly, β‐diversity bacterial community structure changed significantly with ADD and were explained using environmental parameters and inferred functional pathways. Models developed using 24 rib and 34 scapula family‐level taxa allowed the prediction of PMSI with root mean square error of 522.97 ADD (~57 days) and 333.8 ADD (~37 days), respectively.

When deaths occur in water, soft tissue decomposes after a temperature‐dependent period, making DNA identification dependent on bone. This study examined the effects of water on bone DNA purity and quantity, and determined the best of three extraction methods for isolating DNA. The organic phenol–chloroform method consistently extracted DNA approximating the accepted 260/280 purity value (~1.8); ChargeSwitch® gDNA Plant Kit and DNeasy Blood and Tissue Kit produced fair and unacceptable values, respectively. The purity value for humerus and rib samples was consistent across accumulated degree days (ADD). Significant differences in quantification among extraction methods and between bone types were identified. Ribs and ChargeSwitch® gDNA Plant Kit samples produced the lowest mean Ct values of the bone types and the extraction methods, respectively. Therefore, this study proposes that magnetic bead technology extraction methods and ribs be considered when processing bones that have been submerged in water for any length of time.