DNA Analysis Methods Research Articles

Development and validation of a new multiplex for upgrading Y-STRs population databases from 12 to 23 markers and its forensic casework application

Y chromosomal short tandem repeats (Y-STRs) are used in forensic investigations as a useful complementary tool to autosomal markers. The ongoing development of new kits with an increasing number of markers makes it necessary to update populations typed in the Y-STR Haplotype Reference Database to reach at least 23 Y-STRs. A novel Y-STR multiplex panel was developed to offer a cost-efficient alternative to update Y-STR haplotypes from 12 to 23 loci. This panel includes the eleven markers, DYS448, DYS456, DYS458, DYS635, Y-GATA H4, DYS576, DYS481, DYS549, DYS533, DYS570 and DYS643, as well as DYS385a/b for traceability purpose. Developmental validation of this panel was conducted following the recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM), showing high sensitivity, tolerance to common inhibitors as well as high species specificity. It was efficient for degraded DNA samples and for detection of male mixtures. When applying it for extending the current data of the Ibiza population, both the discrimination capacity and the haplotype diversity increased from 0.5952 to 0.9048 and from 0.9808 to 0.9977, respectively. Together, the study demonstrates the suitability of this panel in forensic casework.

A new fluorescence labeling method for molecular analysis of double-stranded DNA

In this study, a double-stranded DNA (dsDNA) fluorescent labeling method was developed using the fusion proteins of fluorescent protein (FP), and 7 kDa DNA-binding family members including Sso7d from Sulfolobus solfataricus, Aho7c from Acidianus hospitalis, ATSV7 from Acidianus tailed spindle virus and Sto7 from Sulfolobus tokodaii. Using this fluorescent DNA labeling method, we succeeded in single-molecule imaging of bacteriophage λDNA molecules stretched on glass surfaces. The fluorescence of the λDNA with FP fusion proteins decayed 2.4- to 6.4-fold slower than that of the typical intercalating method with SYTOX Green (SxG). In addition, the dynamic behaviors of FP-fused Aho7c-λDNA were relaxed and stretched with and without buffer flow, respectively, in microflow channels and were similar to that with typical intercalating dye, such as YOYO-1 and SxG. this fluorescent DNA labeling method. This fluorescent DNA labeling method can solve the problem of rapid fluorescence decay due to the intercalating dyes and therefore can be expected as an alternative to compound-based fluorescent dye. Thus, this study establishes FP fusion proteins as useful fluorescent DNA probes at the single-molecule level.

Developmental validation of the EZ1&2™ DNA Investigator® Kit on the EZ2® Connect Fx

The EZ1&2 DNA Investigator Kit allows automated purification of total DNA from forensic and human identity samples on the EZ2 Connect Fx instrument. Its proven magnetic-bead technology provides high-quality DNA, suitable for direct use in sensitive downstream applications such as quantitative PCR, STR analysis and NGS. The purified DNA obtained is free of proteins, nucleases and inhibitors. In this study, the performance of the EZ1&2 DNA Investigator Kit on the EZ2 Connect Fx was evaluated using typical casework sample types and conditions commonly encountered in forensic and parentage laboratories. Wherever applicable, this validation study followed the recommendations of the European Network of Forensic Science Institutes (ENFSI) and the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM).

Study of pig dna analysis methods in cosmetics: a review

Collagen is one of the components used for cosmetics. Functionally, collagen is derived from pig fat, which functions as a skin conditioning agent (Emollient), emulsion stabilizer (Emulsifier), and as ingredient to increase the viscosity of cosmetic preparations. The method that can detect pig content is the PCR method, which can confirm the presence of DNA content in pigs. In this study, an analysis of cosmetic products containing porcine DNA was carried out using the PCR method. This research was divided into two stages: the initial stage was DNA extraction, and the second stage was PCR analysis. This research is a study through literature study using the narrative review technique. This study aimed to collect information and examine the analysis method of identifying pig DNA contained in cosmetics to obtain the right and accurate way through a literature approach. From the literature study results, it can be concluded that the best extraction method for the isolation of porcine DNA in cosmetic preparations is the extraction method with the Boom Method, while the DNA analysis used to identify porcine DNA in cosmetics is RT-PCR (Real-time Polymerase Chain Reaction). Conventional PCR is because both methods have their respective advantages. The RT-PCR (Real-time Polymerase Chain Reaction) method can amplify and quantify the number of target DNA molecules. The Conventional PCR Method can form DNA bands based on the amplification value.

Developmental validation of STRmix™ NGS, a probabilistic genotyping tool for the interpretation of autosomal STRs from forensic profiles generated using NGS

We describe the developmental validation of the probabilistic genotyping software – STRmix™ NGS – developed for the interpretation of forensic DNA profiles containing autosomal STRs generated using next generation sequencing (NGS) also known as massively parallel sequencing (MPS) technologies. Developmental validation was carried out in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) Guidelines for the Validation of Probabilistic Genotyping Systems and the International Society for Forensic Genetics (ISFG) recommendations and included sensitivity and specificity testing, accuracy, precision, and the interpretation of case-types samples. The results of developmental validation demonstrate the appropriateness of the software for the interpretation of profiles developed using NGS technology.

Optimization and Developmental Validation of 38-plex InDel Panel for Ancestry Inference.

The previously established 38-plex InDel system was optimized and its performance was validated according to the Scientific Working Group on DNA Analysis Method (SWGDAM) application guidelines. The ancestry inference accuracy of individuals from East Asian, European, African and mixed populations was verified. DNA standard sample 9947A was used as the template to establish the optimal amplification conditions by adjusting primer balance, Mg2+ final concentration and optimizing PCR thermal cycle parameters and amplification volume. The allelic dropout, nonspecific amplification and whether the origin of the inferred samples matched the known information were compared to evaluate the performance of this system. The optimal dosage of this system was 0.125-2 ng DNA template. The results of InDel typing were accurate, the amplification equilibrium was good, and the species specificity was good. This system showed certain tolerance to DNA samples including the inhibitor such as hemoglobin (≤80 μmol/L), indigo (≤40 mmol/L), calcium ion (≤1.0 mmol/L), and humic acid (≤90 ng/μL). The system enabled the direct amplification of DNA from saliva and blood on filter paper, and the results of ethnic inference were accurate. The system successfully detected the mixed DNA sample from two individuals. The test results of the system for common biological materials in practical cases were accurate. The results of the 38-plex InDel system are accurate and reliable, and the performance of the system meets the requirement of the SWGDAM guidelines. This system can accurately differentiate the ancestry origins of individuals from African, European, East Asian, and Eurasian populations and can be implemented in forensic practice.

Robust detection of cow and female buffalo DNA through Real-Time PCR assay

Cow, Bos taurus, and female buffalo, Bubalus bubalis, are considered sacred animals that are a part of rural livelihood in India. The purity of products from these bovine species has significant sentimental implications in the dairy and meat industry. Therefore, the mitochondrial DNA and the sex origin, targeting the X and Y chromosomes from these bovine species, were selected to design three multiplex real-time probe PCR assays: Hi-PCR® Cow Detection Kit (MBPCR184), Hi-PCR® Buffalo Detection Kit (MBPCR185) and Hi-PCR® Cattle Sex Determination Kit (MBPCR186). Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines were followed to perform different studies using reference control DNAs. An Internal Reagent Control (IRC) was part of every assay, thus ensuring a successful reaction. The assays were 100% specific, with no cross-amplification of the two bovine species. The amplification of the X chromosomal target was observed for male and female DNAs, whereas Y chromosome amplification was observed only for the male DNA. The assays were 100% specific to the target genes in these organisms with no non-specificity towards any other targets or organisms. The limit of detection for sex determination was 0.01 ng/µl, whereas the differential capability of the assay was 3 copies/µl and 30 copies/µl for Bos taurus and Bubalus bubalis, respectively. The assays were reproducible at 1 ng/µl genomic DNA with 95% CI. The assays are open and compatible with other brands of Real-Time PCR systems used in forensic labs. The experiments presented here verify that the developed real-time PCR assays are robust, produce reliable and reproducible results for detection and differentiation of Bos taurus and Bubalus bubalis and their sex even at low DNA concentrations.

Population genetic data for 17 non-CODIS STR loci for the Saudi Arabian population using the SureID®23comp Human Identification Kit

Our previous work focused on validation the SureID 23comp Human Identification Kit (Health Gene Technologies, China), following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples from the population of Saudi Arabia. The kit genotypes 22 STRs, 17 of which are non-CODIS, and Amelogenin. The validation tests showed that it has the potential to increase the power of testing in complex cases.In this paper, the allele frequency data, common forensic parameters for the 17 non-CODIS STR loci are presented. We found the majority of loci had an excess of homozygosity in the data set, which is most likely explained by the relatively high levels of consanguinity in the population of Saudi Arabia.

Visualization Methods of Latent Fingerprints on Metal Substrates/Cartridges

Despite the development of DNA analysis methods, fingerprint comparisons are still the most common and credible way to identify people when clarifying crimes and other forensically relevant events. Visualization of fingerprints applied to a metal surface, which may be curved (most often cartridges), often represents a difficult task, so that new methods are constantly being developed to get a high-quality visibility of fingerprints. This work describes the most important methods of visualization of fingerprints applied on metal substrates and especially on cartridge cases. So far, there is no suitable technique for making fingerprints visible on fired cartridges. Gun blue techniques, combined techniques with cyanoacrylate and, more recently, electrochemical deposition of polymer films potentiostatically or by cyclic voltammetry, which are fast, cheap and usable in the field, represent a promi­sing way.

Comparison of operational DNA recovery methods: Swabs versus tapelifts

It is routine among many jurisdictions to recover DNA using tapelifts on porous substrates (e.g. clothing) and swabs on non-porous substrates (e.g. tool handles). Here, we examine this by comparing the efficiency of the NSW jurisdiction’s specific swabbing and tapelift techniques on a range of porous and non-porous substrates. To test DNA recovery efficiency, 30 μl aliquots of 1:50 and 1:100 saliva dilutions were deposited onto the substrates, left to dry overnight, recovered, extracted, quantified and a subset profiled. Tapelifts recovered more DNA and DNA profiles with more detectable alleles than swabs for both saliva dilutions on porous substrates. For non-porous substrates, similar DNA quantities and profiles were generally recovered with both methods for both saliva dilutions. These data underpin current practices to recover DNA using tapelifts for porous substrates and swabs for non-porous substrates. These data also revealed severe degradation of DNA recovered from brass, supporting the on-going need to improve DNA recovery and analysis methods for brass substrates.

Development and validation of a new multiplex Y-STR panel designed to increase the power of discrimination.

In an attempt to increase the discrimination capacity (DC) and reduce the adventitious match probability, a 6-dye multiplex Y-chromosomal short tandem repeat (Y-STR) panel named Y34plex was constructed that combined 25 Y-chromosomal markers (DYS456, DYS627, DYS390, DYS570, DYS635, DYS385a/b, DYS448, DYS437, DYS533, DYS449, DYS481, DYS392, DYS391, DYS389I, DYS460, YGATAH4, DYS438, DYS389II, DYS19, DYS458, DYF387S1a/b, DYS439, DYS393, DYS576, and DYS518) in widely used commercial kits, with nine highly polymorphic Y-STR loci (DYS557, DYS527a/b, DYS593, DYS444, DYS596, DYS643, DYS447, DYS549, and DYS645). The Y34plex is a promising type system to distinguish both unrelated and related male individuals due to the incorporation of rapidly mutatedY-STR loci. A validation study of the Y34plex was performed and followed the guidelines of the Scientific Working Group on DNA analysis methods. Results show that full Y-STR profiles were obtained from male/female DNA mixtures with 125pg of male DNA in the presence of 50ng of female DNA. The ability to tolerate polymerase chain reaction inhibitors commonly contained in forensic casework samples demonstrated the applicability and robustness of the Y34plex. Compared with the Yfiler Plus kit, the novel panel showed an increased power of discrimination in Chinese Wuxi Han population (n=434). The overall haplotype diversity of the Y34plex was 0.999606, whereas DC value was 0.956221, which is suitable for use on forensic paternal investigation.

Independent development and validation of a novel six-color fluorescence multiplex panel including 61 diallelic DIPs and 2 miniSTRs for forensic degradation sample.

Current forensic DNA profiles are obtained based on analyses of PCR product sizes or DNA sequence polymorphisms. Sometimes routine forensic analysis using short tandem repeat (STR) generates unsuccessful DNA testing result if the biological sample encountered is excessively degraded and low-template DNA. Herein, a new six-color fluorescence labeling system, including 59 autosomal diallelic deletion or insertion polymorphisms (DIPs), 2 miniSTRs, 2 Y-chromosome DIPs, and 1 Amelogenin gene with the amplicon sizes of less than 200bp, was self-developed. According to the validation guidelines for DNA analysis methods formulated by the Scientific Working Group on DNA Analysis Methods, the validation studies have also been carried out for the multiplex system. This novel panel possessed the features of strong stability, high sensitivity, and good specificity, which was especially suitable for the forensic degraded and mixed sample detections. The cumulative power of exclusion and cumulative matching probability of the system were 0.9999978 and 9.833E-28, respectively, in Han Chinese in Hunan, China. Moreover, this system will be an effective new tool that can be independently applied to forensic personal identification and paternity testing in the populations from the East Asia region, even from the South Asia, America, and Europe regions. The system can also contribute to population phylogenetic affinity and genetic structure analyses among different populations.

Validation of the AGCU Expressmarker 20 + 20Y kit: A 6-dye multiplex assay for forensic application

The AGCU Expressmarker 20 + 20Y Kit is a newly devised short tandem repeat (STR) multiplex system that simultaneously analyses a set of 19 autosomal STR loci, 20 Y chromosomal STR loci and the amelogenin locus with six-dye fluorescent labelling. Here, the AGCU Expressmarker 20 + 20Y system was validated following the guidelines published by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including PCR-condition, sensitivity, mixtures, species specificity, models of inhibition, precision, stutter percentage, concordance, population genetic studies, performance on three kinds of degraded DNA and a type of casework samples. The results indicated that the kit had high sensitivity when there was a small amount of DNA (0.0625 ng), more than one male (minor: major=1:19), or a mixture of males and females (male: female=1:32), models of inhibition (250 μM hematin, 1500 ng/μL humic acid and more than 100 ng/μL tannic acid) and degraded samples. The kit showed high precision level with standard deviation of allele size no more than 0.0930. Furthermore, this system was also tested in 444 random male samples of Chinese Han and Hui population, showing its high discrimination capability in Han and Hui population. Meanwhile, the system was applicable to the case of the AMELY abnormality. In short, the kit was verified and proved to be a robust, reliable and suitable tool for human identification and casework samples.

Technical note: A protein analysis-based method for identifying shahtoosh

Shahtoosh, the down hair of the Tibetan antelope (Pantholops hodgsonii), is a kind of luxury fibre famous for its fineness and softness. Commercial poaching of the animal for its fibre has resulted in a dramatic decline in its population. Microscopic analysis is the most frequently used method for distinguishing shahtoosh, while the PCR-based DNA analysis method is a relatively objective approach. Here, we developed an MALDI-TOF-MS-based protein analysis method for shahtoosh fibre identification. Protein in animal fibres was lysed by SDS/dithiothreitol (DTT)/phosphate buffer. SDS–PAGE was employed to separate and purify the targeted protein. The protein in the gel was enzymatically digested by trypsin and then analysed by MALDI-TOF MS spectrometry. A species-specific peptide marker at 2606 m/z was found for shahtoosh identification. A fibre mixture of 5% shahtoosh mixed with cashmere can be detected with this method. Validation experiments with mixed dyed samples confirmed its ability to detect specific qualitative and quantitative markers. We hope that the method can be widely applied to inspections of shahtoosh and its processed products to minimize the illegal trading of shahtoosh.

Optimized pretreatment conditions for the environmental DNA (eDNA) analysis of Apostichopus japonicus

A non-destructive environmental DNA protocol for the genetic analysis of sea cucumber (Apostichopus japonicus) resources DNA was established. Among the several commercial DNA extraction kits, the DNeasy® Plant Mini Kit was selected as the best choice to obtain the high-quality genomic DNAs from the mucous sea cucumber. As the temperature and incubation time increased, the amount of extracted environmental DNA was also large, but it was judged that the increased amount did not affect as much as 2–3 times. Therefore, these conditions were not considered to be the main factors to consider in actual environmental DNA extraction. However, the amount of seawater relative to the size of the sample was judged as a major consideration, and a sufficient amount of environmental DNA for analysis was secured when stored within 1 min while stirring the volume of seawater corresponding to the total sea cucumber weight (g). In securing the environmental DNA of sea cucumbers, the mortality rate of sea cucumbers in all experiments was 0, and it was judged that the effects of sea cucumbers were not significant through this treatment. Through the results of this study, sea cucumber DNA research, which has been conducted in a destructive method, can be conducted non-destructively through environmental DNA analysis. Through this study, we have secured a standard protocol that can successfully extract the sea cucumber DNA through environmental DNA. It is not only excellent in terms of time and cost of traditional DNA analysis method currently used, but it is completely non-destructive in the ecosystem of the survey area. It is believed that the system can be transformed in a way that does not affect it. However, it is thought that various standard protocols should be established considering the characteristics of each type.

Recommendations for Sustainable Ancient DNA Research in the Global South: Voices From a New Generation of Paleogenomicists.

Paleogenomics - the study of ancient genomes - has made significant contributions, especially to our understanding of the evolutionary history of humans. This knowledge influx has been a direct result of the coupling of next-generation sequencing with improved methods for DNA recovery and analysis of ancient samples. The appeal of ancient DNA studies in the popular media coupled with the trend for such work to be published in “high impact” journals has driven the amassing of ancestral human remains from global collections, often with limited to no engagement or involvement of local researchers and communities. This practice in the paleogenomics literature has led to limited representation of researchers from the Global South at the research design and subsequent stages. Additionally, Indigenous and descendant communities are often alienated from popular and academic narratives that both involve and impact them, sometimes adversely. While some countries have safeguards against ‘helicopter science’, such as federally regulated measures to protect their biocultural heritage, there is variable oversight in others with regard to sampling and exportation of human remains for destructive research, and differing requirements for accountability or consultation with local researchers and communities. These disparities reveal stark contrasts and gaps in regional policies that lend themselves to persistent colonial practices. While essential critiques and conversations in this sphere are taking place, these are primarily guided through the lens of US-based heritage legislation such as the Native American Graves and Protection Act (NAGPRA). In this article, we aim to expand the scope of ongoing conversations by taking into account diverse regional contexts and challenges drawing from our own research experiences in the field of paleogenomics. We emphasize that true collaborations involve knowledge sharing, capacity building, mutual respect, and equitable participation, all of which take time and the implementation of sustainable research methods; amass-and-publish strategy is simply incompatible with this ethos.

Detection the eDNA of Batrachuperus taibaiensis from the Zhouzhi Heihe River Using a Nested PCR Method and DNA Barcoding.

Simple SummaryThe Taibai stream salamander (Batrachuperus taibaiensis) is a protected species endemic to the Zhouzhi Heihe River. Few traditional studies have been conducted on the distribution of B. taibaiensis, leading to irregular discoveries. Environmental DNA (eDNA) is an ideal species detection technique that can increase accuracy and decrease the cost of population surveys. Here, we have established an optimal method for obtaining the eDNA of B. taibaiensis from water samples, which provides a theoretical basis and reference for resource investigation and protection of B. taibaiensis. Moreover, this study highlights the detection of a rare species in a river for use in further research.The Taibai stream salamander (Batrachuperus taibaiensis) is a recently described species of the genus Batrachuperus that occurs in the Zhouzhi Heihe River and is endangered in its native range. Here, we have established a method for water environmental DNA (eDNA) analysis of Batrachuperus using a series of optimizations. We have designed a specific set of primers for the genus Batrachuperus to amplify a 160 bp fragment of Cytb. The sequences were obtained from nested PCR on eDNA extracted from water samples, after which DNA barcoding was performed according to sequence analysis to determine the presence of the target species in the water. The method was validated using water from the Zhouzhi Heihe River with known B. taibaiensis populations and found that B. taibaiensis eDNA can move at least 150 m downstream from its point of origin. This study is the first to establish an optimal method for obtaining the eDNA of Batrachuperus from water samples, which provides a theoretical basis for resource investigation and the protection of B. taibaiensis in future research. It is also an example of the eDNA extraction of other species that live in similar waters and are less genetically diverse between species.

Assessment of human nuclear and mitochondrial DNA qPCR assays for quantification accuracy utilizing NIST SRM 2372a

In forensic DNA casework, a highly accurate real-time quantitative polymerase chain reaction (qPCR) assay is recommended per the Scientific Working Group on DNA Analysis Methods (SWGDAM) (SWGDAM Validation Guidelines for DNA Analysis Methods [1]) to determine whether a DNA sample is of sufficient quantity and robust quality to move forward with downstream short tandem repeats (STR) or sequencing analyses. Most of these assays rely on a standard curve, referred to herein and traditionally as absolute qPCR, in which an unknown is compared, relative to that curve. However, one fundamental issue with absolute qPCR is the quantifiable concentration of commercial assay standards can vary depending on (1) origin, i.e., whether from a cell line or a human subject, (2) supplier, (3) lot number, (4) shipping method, etc. In 2018, the National Institute for Standards and Technology (NIST) released a human DNA standard reference material for evaluating qPCR quantification standards, Standard Reference Material (SRM) 2372a, Romsos et al. (2018) [2] which contains three well-characterized human genomic DNA samples: Component A) a single male1 donor, Component B) a single female1 donor, and Component C) a 1:3 male2:female2 donor, each with certification data for nDNA and informational mitochondrial DNA(mtDNA)/nuclear DNA (nDNA) ratio data. The SRM 2372a was used to assess four qPCR assays: (1) Quantifiler Trio (Thermo Fisher Scientific, Waltham, MA) for nDNA quantification, (2) NovaQUANT (EMD Millipore Corporation, San Diego, CA) for nDNA and mtDNA quantification, (3) a custom duplex mtDNA assay, and (4) a custom triplex mtDNA assay. Additionally, extracts from eighteen (18) skeletal remains were tested with the latter three assays for concordance of DNA concentration and with assays (2) and (3), for the degradation state. Our assessment revealed that an accurate, efficient, and reproducible qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the qPCR chemistry, and (3) the specific primers, and probes (if applicable), used in an assay. Our findings indicate qPCR assays may not always quantify as expected and that performance of each lot should be verified using a well-characterized DNA standard such as the NIST SRM 2372a and adjusted if warranted.

Evaluation of tetrahydrocannabinolic acid (THCA) synthase polymorphisms for distinguishing between marijuana and hemp.

The Controlled Substances Act (CSA) classifies marijuana (Cannabis sativa) as a Schedule I illicit drug. However, the recent Agriculture Improvement Act of 2018 (U.S. Farm Bill) removed hemp from the definition of marijuana in the CSA, making it a legal crop. As a result, many hemp products are now available, including strains of hemp buds high in other cannabinoids such as cannabidiol (CBD) or cannabigerol (CBG). The genetic inheritance of chemical phenotype (chemotype) has been widely studied, with the tetrahydrocannabinolic acid (THCA) synthase gene at the forefront. Previous studies have speculated that there are two forms of the THCA gene, one that produces an active enzyme (present in marijuana) and one that cannot produce a functional enzyme (present in hemp). A DNA analysis method is desirable for determining crop type in sample types inconducive to chemical analysis, such as immature crops, trace residues, small leaf fragments, seeds, and root material. This study optimized and evaluated a previously reported single nucleotide polymorphism (SNP) assay for determining C.sativa crop type. Furthermore, the presence or absence of 15 cannabinoids, including THC and THCA, was reported in cannabis reference materials and 15 legal hemp flower samples. The SNP assay correctly identified crop type in most samples. However, several marijuana samples were classified as hemp, and several hemp seeds were classified as marijuana. Two strains of legal CBG hemp flowers were also classified as marijuana, indicating that factors other than the genetic variation of the THCA synthase gene should be considered when determining crop type.

Alternative direct-to-amplification sperm cell lysis techniques for sexual assault sample processing.

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.