Forensic Body Fluid Identification Using Microbiome Signature Attribution

As submitted by the proposer: Detection and identification of body fluids plays a crucial role in criminal investigation, as it provides information on source of the DNA as well as corroborative evidence regarding the crime committed, scene, and/or association with persons of interest. Historically, casework methods in serology have been chemical, immunological, catalytic, spectroscopic, and/or microscopic in nature. However, most of these methods are presumptive, with few absolutely robust confirmatory exceptions. More importantly, these methods can only be used to identify a single biological fluid, and thus elimination of the presence of other biological fluids and mixtures in forensic samples can be problematic. In recent years several new molecular-based methods (mRNA, miRNA, DNA methylation etc.) have been proposed; although promising, these methods require high quality human DNA or RNA. We instead propose utilization of the known, unique microbial signatures found in these biological fluids. The Human microbiome project reports that the average human harbors 10-100 trillion microbes in or on the body; thus, more than 90% of the cells in or on the human body are of non-human origin. Herein, we propose to utilize these non-human origin cells (specifically bacteria) to develop a single molecular test for the identification of the most forensically relevant human body fluids that could be easily added into a high-throughput sequencing (HTS) forensic panel. We will perform 16S ribosomal DNA (rDNA) high-throughput sequencing on 1400 body fluid samples (200 samples each from venous blood, saliva, semen, vaginal and menstrual secretions, urine and fecal matter) from donors of various ages, ethnicities and genders. Sequence data will be analyzed and statistical analyses of datasets will be used to identify stable core bacteria associated with each biological fluid. After successful identification of the characteristic bacterial signatures for each biological fluid, we will validate the analysis method using 350 blinded body fluid samples in order to show proof of effective identification and to assess potential error. Evaluation of degraded and compromised samples, as well as a panel of mixed body fluid samples will be used to evaluate robustness of the method. Upon successful completion of the research, we will develop and publish a standard operating procedure for collection, preservation, analysis, and interpretation of results. We will be able to provide an economic assessment of associated costs of using these techniques if they were to be integrated into a forensic high-throughput analysis panel. Note: This project contains a research and/or development component, as defined in applicable law. ca/ncf