Author ORCID Identifier
0000-0003-1353-6034
Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Integrative Life Sciences
First Advisor
Tracey Dawson Green
Second Advisor
Edward Boone
Third Advisor
Sarah Seashols-Williams
Fourth Advisor
Anh Bui
Fifth Advisor
Katherine Gettings
Abstract
Analysis of evidentiary samples containing DNA from multiple contributors (“mixtures”) is a time intensive process for a forensic analyst and one where the contributor nature of a sample is not revealed until the end of the traditional forensic workflow. Often, at this stage, retesting or additional testing of mixture samples may not be possible, particularly if the DNA collection device did not preserve the DNA well enough; consequently leaving only trace amounts of a contributor’s DNA present. Thus, a new collection device that would allow for the increased preservation/integrity of evidentiary samples as well as a method that would allow for the subsequent quick and accurate, identification of single source (versus mixture) samples prior to the end-point of STR analysis would be beneficial. The work detailed within has assessed the Bode BioSafe® swab which contains a desiccant and a proprietary compound that prevents degradation, allowing for increased preservation of low-level contributors. Further work towards an improved workflow for these low-level contributors has focused on the development of an integrated qPCR-high resolution melt (HRM) screening assay. This assay would be designed to provide early identification of single source vs. mixed biological evidence samples, pre-STR amplification, at the DNA quantification stage. The developed integrated quantification-HRM mixture screening assay adds two new amplification targets (STR loci D5S818 and D18S51) and an intercalating dye into two existing commercial human DNA quantification chemistries, the Investigator Quantiplex® kit and Quantifiler™ Trio kit, which is then used in combination with added transition and HRM steps post-amplification on the QuantStudio™ 6 Flex. Subsequently, HRM data is imported into a statistical learning tool (scripted in RStudio®/R software) for either a single source prediction or an identification of a “mixture”, allowing for those samples with more than one DNA contributor to be distinguished from those that are single source early in the traditional DNA workflow.
The developed integrated Quantiplex®-HRM assay and Quantifiler™ Trio-HRM assays increase the qPCR reaction volumes without impeding the precision or quality of the resulting human DNA quantification data. For assessment of the developed assays, ~170 single source reference samples and ~32 two-person mixture samples were tested using both linear discriminant analysis (LDA) and support vector machine (SVM) algorithms in R Studio software. For proof-of-concept, only 8 different genotypes, including a genotype of “mixture”, were represented (for each locus) in testing. The developed integrated Quantiplex®-HRM assay and Quantifiler™ Trio-HRM assay have proven to be able to accurately predict the contributor nature of biological samples with 87.9% and 75.0% prediction accuracy, respectively. In addition to accurately predicting samples with the limited genotypes selected for training the machine learning models, the integrated Quantifiler™ Trio-HRM assay has demonstrated the ability to accurately classify samples as a single source or mixture for samples with genotypes not used and/or included in the training standards with 82.5% prediction accuracy. Additionally, the integrated Quantifiler™ Trio-HRM assay can be used for accurate prescreening identification of mixtures with up to five different contributors and mixtures with varying contributor ratios down to 1:100. In addition to reference samples, the integrated Quantifiler™ Trio-HRM assay is capable of accurately predicting mock evidentiary-like samples, with 100.0% mixture accuracy thus decreasing the chances of creating artificial mixtures. These proof-of-concept studies included only a limited number of genotypes thus, simulated data was generated for supplemental reference samples with additional genotypes. Genotypes represented in the training set were able to be increased from 7 (actual DNA samples with single source genotypes) to 66 using simulated data. In its entirety, these studies attest that the developed integrated qPCR-HRM assays as-is can provide early identification of single source and mixed biological evidence samples, pre-amplification. Given the additional evaluation of the integrated Quantifiler™ Trio-HRM assay, this analysis method combined with an online tool complete with the final training database will be ready to deploy to forensic labs for testing.
Rights
© Chastyn J. Smith
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
4-26-2024
Included in
Biology Commons, Categorical Data Analysis Commons, Genetics and Genomics Commons, Statistical Models Commons