Doctoral Dissertation Research: Impact of Various Storage Temperatures andTtime on Microbiome of Forensically Relevant Biological Samples

Virginia Commonwealth University, Richmond VA

Investigators

Abstract

The confirmatory identification of forensically relevant body fluids is a fundamental part to forensic investigations as this information can aid investigators in crime scene reconstruction and resolving mixtures such as those often encountered in sexual assault cases. The majority of current body fluid identification methods are presumptive, and the sample consumption is not ideal for forensic casework. For new methods to be accepted into the community, methods should be robust, reliable, sensitive, and confirmatory to each body fluid while producing stable results over time. This study aims to determine how short- and long-term storage conditions of human biological samples impact the microbial community structures associated with these samples, and to determine whether these conditions impact the accuracy of a newly developed microbial signature-based method for the body fluid identification. Biological sample storage regulations were created without microbial evidence in mind, and it is therefore important to determine whether these samples are stable under various storage times and temperatures. This project will 1) look at the microbial signatures of each body fluid to determine whether there are significant differences in their compositions once stored at various temperatures at extended periods of time and 2) determine whether these storage conditions impact the accuracy of the previously developed body fluid identification method. Samples from ten individuals will be stored at four different temperatures (-20°C, 4°C, 25°C, 37°C) for 3 storage periods (30 days, 60 days, 90 days). To evaluate these changes, 16S rDNA dual-index high throughput sequencing will be performed using the MiSeq FGx sequencing platform. Data analysis will follow the mothur MiSeq SOP. The variation in bacterial signatures will be assessed between all temperature/time groups. Ensemble subspace classification and L1-norm regularized classification methods previously developed will be tested using the bacterial structures obtained to determine how storage conditions may impact the classification accuracy. The findings from this study will be useful in streamlining the previously developed body fluid identification method and will provide additional suggestions for standard operating procedures. This is not only useful for the forensic science community but can contribute to stability, standardization, and mobility of microbial research as a whole. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.