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™ 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.

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.

Sample collection and preservation play a crucial role in whether or not viable information will be obtained from a biological sample.When collecting evidentiary samples, the goal is to maximize the potential for successful short tandem repeat (STR) analysis by recovering the greatest amount of useable DNA as possible without causing contamination. Swabbing is a common method for collecting evidentiary samples [1]. Many variables, such as the swab type, substrate upon which the biological material is deposited, swabbing method, and preservation techniques, can affect the amount and quality of DNA present at the time of analysis [2,3]. Standard swabs used for collecting biological samples for forensic casework include different fibers that are wound around a shaft [4].These fibers are typically either cotton, polyester, rayon, or other types such as padded swabs (a sleeve, usually foam, attached to a shaft) and flocked swabs (fibers, usually nylon, attached to a shaft rather than wound around) [4,5]. A 2014 study reported that

No abstract