Author ORCID Identifier

Defense Date


Document Type

Directed Research Project

First Advisor

Baneshwar Singh

Second Advisor

Sarah Seashols-Williams

Third Advisor

Tomasz Arodz

Fourth Advisor

Denise Wohlfahrt


Body fluid identification (BFID) is one of the most important steps in forensic science as it provides important context to how DNA analysis should be performed on biological samples found at the scene of a crime. Many scientific methods on BFID have been developed over the years with respective benefits and flaws. One emerging method in recent years is microbial signature based BFID. This method uses the same DNA extract that is utilized for human DNA profile generation for microbial analysis. Although DNA is extracted from all body fluid samples without any additional steps, extraction of human DNA from semen samples requires use of the common reducing agent 1,4-dietheothriol (DTT). 1,4-dietheothriol breaks disulfide bonds of sperm cell membrane, and hence helps in efficient release of sperm nuclear DNA. However, it is not clear how addition of DTT impacts bacterial community profile. The purpose of this study was to determine whether the inclusion of DTT had any impact on the bacterial communal structure of semen. Human semen samples were extracted in duplicate using the DNA Investigator Kit (Qiagen, USA) with one group of samples extracted with DTT (n=19) and the other group was extracted without the addition of DTT (n=19). Variable region four of 16S ribosomal DNA (16S rDNA) was amplified and sequenced on the Illumina MiSeq FGx sequencing platform using the MiSeq Reagent Kit V2. Quantification of the extracted DNA indicates bacterial DNA yield didn’t differ significantly between two DNA extraction methods. Sequence analysis revealed that bacterial structure also did not differ significantly between the two DNA extraction methods. While relative abundance of a few bacterial genera differed slightly, it was mostly with low abundance taxa and in general, the presence of DTT had no significant effect on the microbial community structure of the semen samples. Overall, it can be concluded that the bacterial signature-based method for BFID will work equally well with DNA that was extracted either with or without DTT.


© The Author(s)

Is Part Of

VCU Master of Science in Forensic Science Directed Research Projects

Date of Submission


Available for download on Monday, May 08, 2023