Dense single nucleotide polymorphism testing revolutionizes scope and degree of certainty for source attribution in forensic investigations.

The field of forensic DNA analysis has experienced significant advancements over the years, such as the advent of DNA fingerprinting, the introduction of the polymerase chain reaction for increased sensitivity, the shift to a primary genetic marker system based on short tandem repeats, and implementation of national DNA databases. Now, the forensics field is poised for another revolution with the advent of dense single nucleotide polymorphisms (SNPs) testing. SNP testing holds the potential to significantly enhance source attribution in forensic cases, particularly those involving low-quantity or low-quality samples. When coupled with genetic genealogy and kinship analysis, it can resolve countless active cases as well as cold cases and cases of unidentified human remains, which are hindered by the limitations of existing forensic capabilities that fail to generate viable investigative leads with DNA. The field of forensic genetic genealogy combined with genome-wide sequencing can associate relatives as distant as the seventh-degree and beyond. By leveraging volunteer-populated databases to locate near and distant relatives, genetic genealogy can effectively narrow the candidates linked to crime scene evidence or aid in determining the identity of human remains. With decreasing DNA sequencing costs and improving sensitivity of detection, forensic genetic genealogy is expanding its capabilities to generate investigative leads from a wide range of biological evidence.

Detection of invisible biological traces in relation to the physicochemical properties of substrates surfaces in forensic casework.

Touch DNA, which can be found at crime scenes, consists of invisible biological traces deposited through a person's skin's contact with an object or another person. Many factors influence touch DNA transfer, including the "destination" substrate's surface. The latter's physicochemical characteristics (wettability, roughness, surface energy, etc.) will impact touch DNA deposition and persistence on a substrate. We selected a representative panel of substrates from objects found at crime scenes (glass, polystyrene, tiles, raw wood, etc.) to investigate the impact of these characteristics on touch DNA deposition and detection. These were shown to impact cell deposition, morphology, retention, and subsequent touch DNA genetic analysis. Interestingly, cell-derived fragments found within keratinocyte cells and fingermarks using in vitro touch DNA models could be successfully detected whichever the substrates' physicochemistry by targeting cellular proteins and carbohydrates for two months, indoors and outdoors. However, swabbing and genetic analyses of such mock traces from different substrates produced informative profiles mainly for substrates with the highest surface free energy and therefore the most hydrophilic. The substrates' intrinsic characteristics need to be considered to better understand both the transfer and persistence of biological traces, as well as their detection and collection, which require an appropriate methodology and sampling device to get informative genetic profiles.

Interpretation and reporting considerations of low-level DNA profiles following MinElute® purification of PowerPlex® 21 amplified products.

The generation of high-quality DNA profiles from trace amounts of DNA continues to be an issue in forensic casework. Several methods have been proposed over the years to increase recovery rates for low input DNA, including purification of PCR products, an increase in PCR cycle numbers and increasing injection time or voltage during electrophoresis. In this study, the characteristics of DNA profiles generated using QIAGEN MinElute® purification of Promega PowerPlex® 21 amplified products for low DNA input samples, ranging from 80 pg down to 4 pg, were evaluated. MinElute® purification was found to be a simple, effective and time efficient method, which can greatly improve the resolution of amplified PCR products, recovering 100% of donor concordant alleles from as little 16 pg of input template DNA and generating sufficient allelic information for interpretation from as low as 4 pg inputs. However, as is commonly observed with low template DNA samples, the results exhibited extensive disparity in the effects of stochastic variation in amplification, including increased heterozygote peak height imbalance, stutter ratios and instances of allelic drop-in and drop-out, both within and between replicates. As such, it is important that the extent and variability of these stochastic effects are appropriately incorporated in the development of robust profile interpretation guidelines for DNA profiles generated from purified PCR products.

The mysteries of DNA preservation in bone: A comparative study of petrous bones and metacarpal epiphyses using ATR-FTIR spectroscopy.

A comparative analysis of 26 petrous bones and epiphyses of metacarpals from the Second World War era revealed no significant differences in DNA yield or success in STR typing. This unexpected parity in DNA preservation between the petrous bone, a renowned source of endogenous DNA in skeletal remains, and the epiphyses of metacarpals, which are porous and susceptible to taphonomic changes, is surprising. In this study, we introduced ATR-FTIR spectroscopy as an approach to unravel the correlation between bone molecular structure and DNA preservation. Metacarpals and petrous bones with same taphonomic history were sampled and prepared for DNA analyses. While one portion of the sample was used for DNA analysis, the other underwent ATR-FTIR spectroscopic examination. The normalized spectra and FTIR indices between the epiphyses of metacarpals and petrous bones were compared. Because the taphonomic history of the remains used is relatively short and stable, the ATR-FTIR spectroscopy unveiled subtle structural differences between the two bone types. Petrous bones exhibited higher mineralization, whereas epiphyses contained more organic matter. The unexpected preservation of DNA in the epiphyses of metacarpals can likely be attributed to the presence of soft tissue remnants within the trabeculae. Here observed differences in the molecular structure of bones indicate there are different mechanisms enabling DNA preservation in skeletal tissues.

Assessing DNA Degradation through Differential Amplification Efficiency of Total Human and Human Male DNA in a Forensic qPCR Assay.

The assessment of degradation is crucial for the analysis of human DNA samples isolated from forensic specimens. Forensic quantitative PCR (qPCR) assays can include multiple targets of varying amplicon size that display differential amplification efficiency, and thus different concentrations, in the presence of degradation. The possibility of deriving information on DNA degradation was evaluated in a forensic qPCR assay not specifically designed to detect DNA fragmentation, the Plexor HY (Promega), by calculating the ratio between the estimated concentrations of autosomal (99 bp) and Y-chromosomal (133 bp) targets ("[Auto]/[Y]"). The [Auto]/[Y] ratio measured in 57 formalin-fixed, paraffin-embedded samples was compared to a quality score (QS) calculated for corresponding STR profiles using quantitative data (allele peak height). A statistically significant inverse correlation was observed between [Auto]/[Y] and QS (R = -0.65, p < 0.001). The [Auto]/[Y] values were highly correlated (R = 0.75, p < 0.001) with the "[Auto]/[D]" values obtained using the PowerQuant (Promega) assay, expressly designed to detect DNA degradation through simultaneous quantification of a short (Auto) and a long (D) autosomal target. These results indicate that it is possible to estimate DNA degradation in male samples through Plexor HY data and suggest an alternative strategy for laboratories lacking the equipment required for the assessment of DNA integrity through dedicated qPCR assays.

Evaluation of post-PCR purification methods and subsequent optimisation of the MinElute® PCR Purification Kit for low input DNA samples amplified with PowerPlex® 21.

Weak and partial DNA profiles are commonly encountered within forensic casework due to amplification of low DNA input samples. One option for increasing allelic detection in such samples is the purification of amplified PCR product using commercially available column-based methods. In this study, four commercially available post-PCR purification methods, QIAGEN MinElute®, Independent Forensics Amplicon™ Rx, Millipore Microcon® and Thermo Fisher Scientific ExoSAP-IT™ were evaluated, comparing the quality of PowerPlex® 21 DNA profiles produced to the standard DNA profile generated prior to purification. An increased detection of alleles above the analytical threshold was observed following purification with the MinElute®, Amplicon™ Rx and Microcon® methods, allowing informative DNA profiles to be recovered using as little as 8 pg DNA. However, post-PCR purification using the ExoSAP-IT™ kit was unsuccessful, with no alleles detected above analytical threshold in samples with ≤16 pg DNA. The MinElute® kit was selected for optimisation on the basis of DNA profile quality, including increased detection of alleles and minimal artefacts. The MinElute® method was optimised by evaluating the number of washes and final elution buffer volume, resulting in a further increase in detection of alleles by reducing the elution buffer volume. Overall, this study showed that PowerPlex® 21 DNA profiles from low input DNA can be successfully enhanced by employing the MinElute® post-PCR purification method.

Recovery of DNA from acetaminophen exploring physical state and sampling methods.

Research into the recovery of DNA from illicit drug samples has shown it is possible to get forensically useful profiles from such substrates. However, it is not yet known if the different physical states that drugs can be found in influences the quantity and quality of DNA that can be recovered or what is the best sampling method to adopt for powdered samples. This research used acetaminophen in four different states - large crystalline, powder, in solution, or residue - to determine the efficacy of current DNA technology in recovery and analysis of the resulting sample. Five replicates of each were prepared. Human blood was deposited on or mixed with the drug and left for 1 hour. The surface of the drug was sampled by wet/dry swabbing (where appropriate), or the entire sample was deposited in a tube, and the DNA then extracted using DNA-IQ™. The amount of DNA recovered (ng), degradation index, number of PCR cycles (Ct) required for the IPC to reach threshold, number of alleles in the DNA profile and average peak height (APH) were assessed. All samples, irrespective of the physical state they were collected from, returned full DNA profiles that corresponded to the DNA profile of the blood donor, with no degradation or inhibition detected. It was also found the wet/dry swabbing method returned higher levels of DNA than inclusion of the entire sample into the tube for powdered acetaminophen and the appropriate method to use will be dependent on casework circumstances. The findings of this research further develops our understanding of the recovery of DNA from drugs, and supports the need for further investigation to understand under what conditions DNA can be recovered from illicit substances.

[Authenticity and adulteration identification of Bubali Cornu based on DNA fingerprints].

Whether adulteration exists is a difficult problem in the identification of traditional Chinese medicine(TCM). Bubali Cornu is mainly available in the medicinal material market in the form of buffalo horn silk or buffalo horn powder but lacks obvious identification characteristics, so there is a risk of adulteration. However, the method of identification of adulteration in Bubali Cornu is lacking at present. In order to ensure authenticity and identify adulteration of TCM Bubali Cornu, control the quality of TCM Bubali Cornu, and ensure the authenticity of clinical use, the DNA fingerprints of 43 batches of samples from pharmaceutical companies and medicinal material markets were identified, and the amplification primers of fluorescent DNA fingerprints of Bubali Cornu and Bovis Grunniens Cornu were screened. The DNA fingerprints of Bubali Cornu were obtained by fluorescent capillary typing. The identification effect of fluorescent capillary typing on different adulteration ratios was also tested. Two pairs of fluorescent STR typing primers, namely 16Sa and CRc, which can distinguish Bubali Cornu and Bovis Grunniens Cornu, were screened out, and a DNA fingerprint identification method was established. The 16Sa migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 223.4-223.9 bp and 225.5-226.1 bp. The CRc migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 518.8-524.8 bp and 535.9-542.5 bp. The peak height of the migration peak could be used for preliminary quantification of the adulterants with an adulteration ratio below 50%, and the quantitative results were similar to the adulteration ratio. In this study, a simple and quick universal DNA fingerprint method was established for the identification of Bubali Cornu and its adulterants, which could realize the identification of TCM Bubali Cornu and the semi-quantitative identification of the adulterants.

PCR in Forensic Science: A Critical Review.

The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.

Biogeographical Ancestry Analyses Using the ForenSeqTM DNA Signature Prep Kit and Multiple Prediction Tools.

The inference of biogeographical ancestry (BGA) can assist in police investigations of serious crime cases and help to identify missing people and victims of mass disasters. In this study, we evaluated the typing performance of 56 ancestry-informative SNPs in 177 samples using the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx system. Furthermore, we compared the prediction accuracy of the tools Universal Analysis Software v1.2 (UAS), the FROG-kb, and GenoGeographer when inferring the ancestry of 503 Europeans, 22 non-Europeans, and 5 individuals with co-ancestry. The kit was highly sensitive with complete aiSNP profiles in samples with as low as 250pg input DNA. However, in line with others, we observed low read depth and occasional drop-out in some SNPs. Therefore, we suggest not using less than the recommended 1ng of input DNA. FROG-kb and GenoGeographer accurately predicted both Europeans (99.6% and 91.8% correct, respectively) and non-Europeans (95.4% and 90.9% correct, respectively). The UAS was highly accurate when predicting Europeans (96.0% correct) but performed poorer when predicting non-Europeans (40.9% correct). None of the tools were able to correctly predict individuals with co-ancestry. Our study demonstrates that the use of multiple prediction tools will increase the prediction accuracy of BGA inference in forensic casework.

A DNA typing panel of 201 genetic markers for degraded samples: development and validation.

With the increasing number of complex forensic cases in recent years, it's more important to combine the different types of genetic markers such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (InDels), and microhaplotypes (MHs) to provide more genetic information. In this study, we selected totally 201 genetic markers, including 24 autosomes STRs (A-STRs), 24 Y chromosome STRs (Y-STRs), 110 A-SNPs, 24 Y-SNPs, 9 A-InDels, 1 Y-InDel, 8 MHs, and Amelogenin to establish the HID_AM Panel v1.0, a Next-Generation Sequencing (NGS) detection system. According to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the repeatability, accuracy, sensitivity, suitability for degraded samples, species specificity, and inhibitor resistance of this system were assessed. The typing results on 48 STRs and Amelogenin of this system were completely consistent with those obtained using capillary electrophoresis. This system accurately detected 79 SNPs as parallelly confirmed by a FGx sequencer with the ForenSeq™ DNA Signature Prep Kit. Complete allele typing results could be obtained with a DNA input of no less than 200 pg. The detection success rate of this system was significantly higher than that of the GlobalFiler™ kit when the degradation index of mock degraded sample was greater than 15.87. When the concentration of hematin in the amplification system was ≤40 µmol/L, indigo blue was ≤2 mmol/L, or humic acid was ≤15 ng/µL, amplification was not significantly inhibited. The system barely amplified the DNA extract from duck, mouse, cow, rabbit, and chick. The detection rate of STRs on routine samples of this panel is 99.74%, while all the SNPs, InDels, and MHs were successfully detected. In summary, we setup a NGS individual typing panel including 201 genetic markers with the high accuracy, sensitivity, species specificity, and inhibitors resistance, which is applicable for individual identification of degraded samples.

Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer.

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

Evaluation of the usefulness of insertion-null markers in critical skeletal remains.

Forensic DNA analysis in compromised skeletal remains may pose challenges due to DNA degradation, often resulting in partial or negative autosomal STRs profiles. To address this issue, alternative approaches such as mitochondrial DNA or SNPs typing may be employed; however, they are labour-intensive and costly. Insertion-null alleles (INNULs), short interspersed nuclear elements, have been suggested as a valuable tool for human identification in challenging samples due to their small amplicon size. A commercial kit including 20 INNULs markers along with amelogenin (InnoTyper® 21) has been developed. This study assesses its utility using degraded skeletal remains, comparing the results obtained (the number of detected alleles, RFU values, PHR, and the number of reportable markers) to those obtained using GlobalFiler™. Subsequently, the random match probability of the two profiles for each sample was determined using Familias version 3 to evaluate the power of discrimination of the results obtained from each kit. In every sample, InnoTyper® 21 yielded more alleles, higher RFU values, and a greater number of reportable loci. However, in most cases, both profiles were similarly informative. In conclusion, InnoTyper® 21 serves as a valuable complement to the analysis of challenging samples in cases where a poor or negative profile was obtained.

Evaluation of metal ions and DNA recovery from the surface of fired and unfired brass ammunition to improve STR profiling.

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence.

Analysis of 26 STR loci (PowerPlex® Fusion 6C System) in a mestizo population from Mexico city.

BACKGROUND: Short tandem repeats (STRs) are the most widely used genetic markers in forensic genetics. Therefore, it is essential to document genetic population data of new kits designed for human identification purposes to enable laboratories to use these genetic systems to interpret and solve forensic casework. However, in Mexico, there are no studies with the PowerPlex Fusion 6C System, which includes 26 STRs (23 autosomal STRs and 3 Y-STRs). METHODS AND RESULTS: 600 DNA samples from Mexico City were subjected to genotyping using the PowerPlex Fusion 6C System. For autosomal STRs, 312 different alleles were observed. Combined PE and PD were 99.999999809866% and 99.99999999999999999999999818795%, respectively. Genetic distances and AMOVA test showed low but significant differentiation between Mexican populations. CONCLUSIONS: The results reported in this work demonstrate the efficacy of this system for human identification purposes in the population studied and justify its possible application in other Mexican Mestizo populations.

Technical Note: A simple FTA® based method for the direct STR amplification of human foetal tissues.

Short tandem repeats (STRs) or microsatellites are short, tandemly repeated DNA sequences that involve a repetitive unit of 1-6 bp. DNA isolation and purification from a large number and often compromised samples gives problems to forensic labs for STR typing. Many of the conventional methods used in the isolation and purification of DNA from forensic samples are time consuming, expensive, hazardous for health and are often associated with greater risks of cross contamination. FTA® technology is a method designed to simplify the collection, shipment, archiving and purification of nucleic acid from a wide variety of biological samples. We report a new method for the direct STR amplification which can amplify STR loci from human foetal tissues spotted on FTA cards, bye-passing the need of DNA purification. The STR loci amplified by this method was compared with conventional method of STR profiling and was found absolutely matching. Therefore, this new method is demonstrated to be very useful for fast, less expensive and non- hazardous forensic DNA analysis.

A preliminary study on identification of the blood donor in a body fluid mixture using a novel compound genetic marker blood-specific methylation-microhaplotype.

Blood-containing mixtures are frequently encountered at crime scenes involving violence and murder. However, the presence of blood, and the association of blood with a specific donor within these mixtures present significant challenges in forensic analysis. In light of these challenges, this study sought to address these issues by leveraging blood-specific methylation sites and closely linked microhaplotype sites, proposing a novel composite genetic marker known as "blood-specific methylation-microhaplotype". This marker was designed to the detection of blood and the determination of blood donor within blood-containing mixtures. According to the selection criteria mentioned in the Materials and Methods section, we selected 10 blood-specific methylation-microhaplotype loci for inclusion in this study. Among these loci, eight exhibited blood-specific hypomethylation, while the remaining two displayed blood-specific hypermethylation. Based on data obtained from 124 individual samples in our study, the combined discrimination power (CPD) of these 10 successfully sequenced loci was 0.999999298. The sample allele methylation rate (Ram) was obtained from massive parallel sequencing (MPS), which was defined as the proportion of methylated reads to the total clustered reads that were genotyped to a specific allele. To develop an allele type classification model capable of identifying the presence of blood and the blood donor, we used the Random Forest algorithm. This model was trained and evaluated using the Ram distribution of individual samples and the Ram distribution of simulated shared alleles. Subsequently, we applied the developed allele type classification model to predict alleles within actual mixtures, trying to exclude non-blood-specific alleles, ultimately allowing us to identify the presence of blood and the blood donor in the blood-containing mixtures. Our findings demonstrate that these blood-specific methylation-microhaplotype loci have the capability to not only detect the presence of blood but also accurately associate blood with the true donor in blood-containing mixtures with the mixing ratios of 1:29, 1:19, 1:9, 1:4, 1:2, 2:1, 7:1, 8:1, 31:1 and 36:1 (blood:non-blood) by DNA mixture interpretation methods. In addition, the presence of blood and the true blood donor could be identified in a mixture containing four body fluids (blood:vaginal fluid:semen:saliva = 1:1:1:1). It is important to note that while these loci exhibit great potential, the impact of allele dropouts and alleles misidentification must be considered when interpreting the results. This is a preliminary study utilising blood-specific methylation-microhaplotype as a complementary tool to other well-established genetic markers (STR, SNP, microhaplotype, etc.) for the analysis in blood-containing mixtures.

Effects and considerations of multiplexing ForenSeq Kintelligence libraries with a negative control.

The negative template control or negative amplification control has been an essential component of the forensic DNA analysis workflow that helps monitor contamination. As such, the inclusion of a negative control in forensic DNA analysis has been a requirement for all laboratories audited under the FBI's Quality Assurance Standards. As massively parallel sequencing (MPS) becomes more conventional in forensic laboratories, considerations for the inclusion of a negative control in every sequencing run can be evaluated. Although the inclusion of a negative control in library preparation and the first sequencing run has a practical function, there is less utility for its inclusion in all subsequent sequencing runs for that library preparation. Although this is universal to all MPS assays, it is most relevant for an assay that has a low sample multiplexing capacity, such as the ForenSeq Kintelligence Kit (Qiagen/Verogen, Inc.). The ForenSeq Kintelligence Kit is an investigative genetic genealogy (IGG) sequencing-based assay that targets 10,230 forensically relevant single-nucleotide polymorphisms. The manufacturer recommends multiplexing 3 libraries per sequencing run, which includes controls. The purpose of this study was to investigate the effect of the inclusion of a negative control in every Kintelligence sequencing run. We observed that the library generated from a negative amplification control will take 7%-14% of the run output. The loss of sequencing space taken by a negative control decreased the available output for DNA-containing samples, leading in some cases to allele or locus dropout and accompanying higher numbers of sixth to seventh order unknown associations in GEDmatch PRO.

A developmental validation of the Quick TargSeq 1.0 integrated system for automated DNA genotyping in forensic science for reference samples.

Analysis of short tandem repeats (STRs) is a global standard method for human identification. Insertion/Deletion polymorphisms (DIPs) can be used for biogeographical ancestry inference. Current DNA typing involves a trained forensic worker operating several specialized instruments in a controlled laboratory environment, which takes 6-8 h. We developed the Quick TargSeq 1.0 integrated system (hereinafter abbreviated to Quick TargSeq) for automated generation of STR and DIP profiles from buccal swab samples and blood stains. The system fully integrates the processes of DNA extraction, polymerase chain reaction (PCR) amplification, and electrophoresis separation using microfluidic biochip technology. Internal validation studies were performed using RTyper 21 or DIP 38 chip cartridges with single-source reference samples according to the Scientific Working Group for DNA Analysis Methods guidelines. These results indicated that the Quick TargSeq system can process reference samples and generate STR or DIP profiles in approximately 2 h, and the profiles were concordant with those determined using traditional STR or DIP analysis methods. Thus, reproducible and concordant DNA profiles were obtained from reference samples. Throughout the study, no lane-to-lane or run-to-run contamination was observed. The Quick TargSeq system produced full profiles from buccal swabs with at least eight swipes, dried blood spot cards with two 2-mm disks, or 10 ng of purified DNA. Potential PCR inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect the amplification reactions of the instrument. The overall success rate and concordance rate of 153 samples were 94.12% and 93.44%, respectively, which is comparable to other commercially available rapid DNA instruments. A blind test initiated by a DNA expert group showed that the system can correctly produce DNA profiles with 97.29% genotype concordance with standard bench-processing methods, and the profiles can be uploaded into the national DNA database. These results demonstrated that the Quick TargSeq system can rapidly generate reliable DNA profiles in an automated manner and has the potential for use in the field and forensic laboratories.

Utilizing Massively Parallel Sequencing (MPS) of Human Leukocyte Antigen (HLA) Gene Polymorphism to Assess Relatedness in Deficiency Parentage Testing.

In the realm of DNA testing with legal implications, the reliability and precision of genetic markers play a pivotal role in confirming or negating paternity claims. This study aimed to assess the potential utility of human leukocyte antigen (HLA) gene polymorphism through massively parallel sequencing (MPS) technology as robust forensic markers for parentage testing involving genetic deficiencies. It sought to redefine the significance of HLA genes in this context. Data on autosomal short tandem repeat (aSTR) mutational events across 18 paternity cases involving 16 commonly employed microsatellite loci were presented. In instances where traditional aSTR analysis failed to establish statistical certainty, kinship determination was pursued via HLA genotyping, encompassing the amplification of 17 linked HLA loci. Within the framework of this investigation, phase-resolved genotypes for HLA genes were meticulously generated, resulting in the definition of 34 inherited HLA haplotypes. An impressive total of 274 unique HLA alleles, which were classified at either the field 3 or 4 level, were identified, including the discovery of four novel HLA alleles. Likelihood ratio (LR) values, which indicated the likelihood of the observed data under a true biological relationship versus no relationship, were subsequently calculated. The analysis of the LR values demonstrated that the HLA genes significantly enhanced kinship determination compared with the aSTR analysis. Combining LR values from aSTR markers and HLA loci yielded conclusive outcomes in duo paternity cases, showcasing the potential of HLA genes and MPS technology for deeper insights and diversity in genetic testing. Comprehensive reference databases and high-resolution HLA typing across diverse populations are essential. Reintegrating HLA alleles into forensic identification complements existing markers, creating a potent method for future forensic analysis.