Jordan O. Cox

In recent years, DNA analysis techniques have drastically increased in sensitivity, allowing for low template DNA samples to be more easily detected and used for identification. Since the problems inherent in low template DNA are exacerbated in DNA mixture samples, it would be advantageous to incorporate an assay earlier in the DNA workflow that could detect a mixture and, potentially, determine the number of contributors. Some real-time PCR instruments have high-resolution melt curve analysis (HRM) capabilities, allowing for an opportunity to integrate an HRM screening assay into a commercial DNA quantification kit. This work describes the integration of a mixture screening HRM assay using STR loci D5S818 and D18S51 into Qiagen’s Investigator Quantiplex^® kit. The integrated Quantiplex^®-HRM assay was tested on two qPCR platforms: The Rotor-Gene^® Q and the QuantStudio™ 6 Flex. Data from this assay was analyzed using linear discriminant and support vector machine analyses for sample classification. When HRM curve data from the Rotor-Gene^® Q was used for classification, the integrated assay exhibited an overall accuracy of 89.39%, correctly classifying 87.5% of single source samples and 100% of mixtures. When HRM curve data from the QuantStudio™ 6 Flex was used for classification, the integrated assay exhibited an overall accuracy of 87.88%, correctly classifying 87.5% of single source samples and 90% of mixtures. The overall accuracy of the integrated Quantiplex^®-HRM assay on both instruments met our goal of ≥ 80% accuracy, demonstrating the viability of the assay to detect mixtures when integrated into a commercial quantification kit.

Despite the improvements in forensic DNA quantification methods that allow for the early detection of low template/challenged DNA samples, complicating stochastic effects are not revealed until the final stage of the DNA analysis workflow. An assay that would provide genotyping information at the earlier stage of quantification would allow examiners to make critical adjustments prior to STR amplification allowing for potentially exclusionary information to be immediately reported. Specifically, qPCR instruments often have dissociation curve and/or high-resolution melt curve (HRM) capabilities; this, coupled with statistical prediction analysis, could provide additional information regarding STR genotypes present. Thus, this study aimed to evaluate Qiagen’s principal component analysis (PCA)-based ScreenClust^® HRM^® software and a linear discriminant analysis (LDA)-based technique for their abilities to accurately predict genotypes and similar groups of genotypes from HRM data. Melt curves from single source samples were generated from STR D5S818 and D18S51 amplicons using a Rotor-Gene^® Q qPCR instrument and EvaGreen^® intercalating dye. When used to predict D5S818 genotypes for unknown samples, LDA analysis outperformed the PCA-based method whether predictions were for individual genotypes (58.92% accuracy) or for geno-groups (81.00% accuracy). However, when a locus with increased heterogeneity was tested (D18S51), PCA-based prediction accuracy rates improved to rates similar to those obtained using LDA (45.10% and 63.46%, respectively). This study provides foundational data documenting the performance of prediction modeling for STR genotyping based on qPCR-HRM data. In order to expand the forensic applicability of this HRM assay, the method could be tested with a more commonly utilized qPCR platform.

The prevalence of sexual assault cases and increasingly sensitive DNA analysis methods have resulted in sexual assault kit backlogs in the United States. Although traditional DNA extraction and purification utilizing detergents, proteinase K, and DTT have been the primary technique for lysing sperm cell fractions from these samples, it is labor-intensive and inefficient regarding time and sperm DNA recovery - hindering the ability of forensic analysts to keep pace with evidence submissions. Thus, this study examined seven alternative sperm cell lysis techniques to develop a method that could efficiently lyse sperm and consistently generate high-quality profiles while also reducing time, labor, and cost requirements. Microscopic examination of lysates indicated only Casework Direct and alkaline techniques could lyse all spermatozoa within samples, while quantification results demonstrated all methods performed comparably to the control method of forensicGEM (TM) Sperm (p > 0.06). Amplification with 0.25 ng DNA revealed that unpurified lysates from Casework Direct, alkaline, and NP-40 techniques produced DNA profiles with acceptable mean STR peak heights and interlocus balance, both of which were similar to or better than the control. Overall, this study demonstrated the ability of Casework Direct, alkaline, and NP-40 methods to efficiently lyse spermatozoa and provide high-quality STR profiles despite the absence of a purification step. Ultimately, based on the data reported herein, alkaline lysis is the recommended alternative sperm lysis approach given its ability to generate high-quality profiles, save time, and decrease the cost per reaction when compared to traditional sperm cell lysis methods.

Sample collection at the crime scene can introduce variations in DNA recovery based upon the substrate from which a sample is collected, the material of the collection device used, or the storage conditions after collection. There are many factors during this process that can degrade the sample during drying and storage, and before DNA extraction can be performed. The purpose of this study was to evaluate and compare the performance of standard cotton swab collection with the Bode BioSafe^® swab, which includes both a desiccant at the swab head and proprietary compounds to prevent degradation of the sample during sample collection and preservation. Blood and touch DNA samples were collected from porous and nonporous substrates and stored at elevated temperatures to simulate accelerated time. DNA quantification and STR profile data were used to assess the performance of the swabs. BioSafe^® swab collection resulted in similar DNA yields from blood samples and significantly higher DNA yields from touch samples when compared to collection with cotton swabs. BioSafe^® swabs also resulted in higher DNA integrity during long‐term storage, increased STR profile success and improved retention of low‐level contributor alleles.

DNA extractions of semen samples commonly utilize dithiothreitol (DTT) to reduce and disrupt disulfide bonds. Although traditional extraction techniques remove DTT before downstream analyses, the forensic DNA community has recently explored Y‐screening, direct amplification, and direct cell lysis assays that omit purification but employ reducing agents to lyse spermatozoa. This study examined the impact of residual DTT on downstream processes involving fluorescent dyes. Quantification using Investigator^® Quantiplex HYres revealed a significant increase in the male DNA yield (p = 0.00056) and a >150,000,000‐fold increase in the male:human DNA ratio when DTT remained in extracts versus when it was filtered out using a traditional purification method. When DTT was present with Quantifiler™ Trio, the true mean DNA yield for the large autosomal target significantly increased (p = 0.038) and the average reported DNA yields increased 1.1‐fold, >9.5‐fold, and 1.3‐fold for the small autosomal, large autosomal, and male targets, respectively. DTT‐spiked DNA standards from both kits were impacted similarly to samples with residual DTT, demonstrating that observed effects were related to DTT and not the extraction method. This study corroborates other reports that DTT adversely affects multiple dyes (e.g., Cy5, Quasar 670, SYBR Green I, TMR, and Mustang Purple^®). Overall, DTT causes inaccurate quantities and, consequently, inaccurate calculated male:female ratios when used in conjunction with these kits. Thus, implementation of newer direct‐to‐PCR assays incorporating DTT should either be avoided or used only with carefully evaluated, compatible dyes.

Due to recent improvements in forensic DNA testing kit sensitivity, there has been an increased demand in the criminal justice community to revisit past convictions or cold cases. Some of these cases have little biological evidence other than touch DNA in the form of archived latent fingerprint lift cards. In this study, a previously developed optimised workflow for this sample type was tested on aged fingerprints to determine if improved short tandem repeat (STR) profiles could be obtained. Two-year-old samples processed with the optimised workflow produced an average of approximately five more STR alleles per profile over the traditional method. The optimised workflow also produced detectable alleles in samples aged out to 28 years. Of the methods tested, the optimised workflow resulted in the most informative profiles from evidence samples more representative of the forensic need. This workflow is recommended for use with archived latent fingerprint samples, regardless of the archival time.

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low‐template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re‐extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low‐template DNA sample processing are not enough to justify the risk associated with additional manipulation.

Sexual assault evidence often contains sperm cells, which are typically separated from nonsperm cells using manual differential lysis procedures. The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment. DNA yields were determined by qPCR, followed by multiplex STR amplification, CE analysis, and mixture interpretation. The automated method was capable of effective cell separation, producing DNA yields sufficient for STR amplification. Further, sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures, with 6–8× fewer female alleles detected (median 1.5 alleles). Ultimately, using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.