Microbial DNA in human DNA extracts: recoverability of the microbiome in DNA extracts stored frozen long-term and its potential and ethical implications for forensic investigation

Human DNA samples can remain unaltered for years and stored DNA extracts from such samples can preserve important genetic information for forensic investigations for long periods of time. Besides human genetic information, these extracts potentially contain an additional source of valuable information: microbiome signatures. Forensic microbiology is rapidly developing to become a significant tool for estimating post-mortem interval (PMI), and establishing cause of death and personal identity. To date, the possibility to recover unaltered microbiome signatures from human DNA extracts has not been proven. This study examines the microbiome signatures within human DNA extracts obtained from six cadavers with different postmortem intervals, which were stored frozen for between 4 and 15 years. The results demonstrate that the microbiome can be co-extracted with human DNA using forensic kits designed to extract the human host’s DNA from different tissues and fluids during different stages of decomposition. We compared the type and number of microbial communities identified in these samples with microbial DNA recovered from two human cadavers donated to the Forensic Anthropology Center at Texas State University (FACTS) during multiple stages of decomposition, to examine whether the microbial signatures recovered from old human DNA extracts are consistent with microbial signatures from recently extracted microbial DNA associated with human decomposition. Microbial DNA was isolated from these control samples using QIAamp PowerFecal Pro DNA Kit. All the DNA extracts (human and microbial) were quantified and the V4 region of 16S rRNA gene was sequenced using Illumina MiSeq platform. All extracts yielded successful amplification of 16S rRNA gene. The results obtained from the human DNA extracts were compared with each other and with the microbial DNA from the FACTS samples. Overall, we found that the presence and recovery of specific microbial taxa depends on the decomposition stage, the type of tissue, and the environment in which the cadaver decomposed. We found no indications of contamination in the microbial signatures, or any alterations that could be attributed to the long-term frozen storage of the human DNA extracts, demonstrating that demonstrate that older human DNA extracts are a good source of such microbial signatures. No shared Core Microbiome (CM) was identified amongst the total 18 samples, but when samples are grouped based on the stage of decomposition, certain species are associated with the different stages, offering potential for the use of microbial signatures co-extracted with human DNA samples for PMI estimation in future. This new significance of older human DNA extracts brings with it important ethical-legal considerations. Currently, there are no shared protocols or legal frameworks governing the long-term storage and use of human DNA extracts obtained from crime scene evidence for additional research purposes. To harness the potential of microbiome analysis of older human DNA extracts, it is important to create shared protocols on the storage of biological material collected at crime scenes. We review existing legislation and guidelines, and identify some important limitations for the further development and application of forensic microbiomics