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Training is an essential component of onboarding new hires in forensic science service provider (FSSP) laboratories. There are several DNA training standards published by the American Academy of Forensic Sciences (AAFS) Academy Standards Board (ASB) American National Standards Institute (ANSI) accredited framework. In this study, we conducted a survey of forensic DNA laboratory training programs to better understand training activities and materials. The survey was approved by the IRB and emailed to forensic laboratory directors, assistant directors, and/or DNA technical leaders and responses were submitted by them or their designees. Over thirty leaders and stakeholders responded. In this article, we report on the results of the survey. Respondents indicated that training activities included readings, writing assignments and quizzes, shadowing analysts, and mock casework and that training is documented and is a collaborative effort of the technical leader, unit supervisor, advanced forensic scientists, and other analysts and technicians. Laboratories assess competency using multiple methods including performance on mock casework, report writing, laboratory practical and competency tests, and a mock trial and testimony. The top three training activities reported are hands‐on practice, shadowing, and readings. The top three focuses of the training are laboratory techniques (extraction, quantitation, amplification, and capillary electrophoresis), STR typing, and mixture analysis. Ethics violations and failure to pass the competency tests and mock trial, even after remediation, are the top reasons new hires fail training. Finally, the top items respondents would like to spend more time offering training on are troubleshooting, validation, and root cause analysis. [ABSTRACT FROM AUTHOR]

Advancements in forensic DNA typing technology and methods have increased sensitivity and, while beneficial, carry the weight of more challenging profile interpretation. In response, the forensic DNA community has often requested more complex reference materials to address commonly encountered measurement and interpretation issues such as complex DNA mixtures, DNA degradation, and PCR inhibition. The National Institute of Standards and Technology (NIST) released Research Grade Test Material 10235: Forensic DNA Typing Resource Samples to support the forensic DNA community. Components include three single source samples, two degraded samples, and three mixture samples. As part of the Research Grade Test Material (RGTM) process, automated methods for bottling, alternative sample tube types, and the addition of carrier RNA for stabilizing low‐quantity samples were investigated. Both internal and external testing demonstrate the stability of the material over time at 4°C through qPCR testing. In the development of a data portal, users have been allowed to anonymously upload results and compare their data with NIST and others. This report describes the preparation and stability of this material. [ABSTRACT FROM AUTHOR]

Genetic assessment of highly incinerated and/or degraded human skeletal material is a persistent challenge in forensic DNA analysis, including identifying victims of mass disasters. Few studies have investigated the impact of thermal degradation on whole‐genome single‐nucleotide polymorphism (SNP) quality and quantity using next‐generation sequencing (NGS). We present whole‐genome SNP data obtained from the bones and teeth of 27 fire victims using two DNA extraction techniques. Extracts were converted to double‐stranded DNA libraries then enriched for whole‐genome SNPs using unpublished biotinylated RNA baits and sequenced on an Illumina NextSeq 550 platform. Raw reads were processed using the EAGER (Efficient Ancient Genome Reconstruction) pipeline, and the SNPs filtered and called using FreeBayes and GATK (v. 3.8). Mixed‐effects modeling of the data suggest that SNP variability and preservation is predominantly determined by skeletal element and burn category, and not by extraction type. Whole‐genome SNP data suggest that selecting long bones, hand and foot bones, and teeth subjected to temperatures <350°C are the most likely sources for higher genomic DNA yields. Furthermore, we observed an inverse correlation between the number of captured SNPs and the extent to which samples were burned, as well as a significant decrease in the total number of SNPs measured for samples subjected to temperatures >350°C. Our data complement previous analyses of burned human remains that compare extraction methods for downstream forensic applications and support the idea of adopting a modified Dabney extraction technique when traditional forensic methods fail to produce DNA yields sufficient for genetic identification. [ABSTRACT FROM AUTHOR]

Massively parallel sequencing allows for integrated genotyping of different types of forensic markers, which reduces DNA consumption, simplifies experimental processes, and provides additional sequence-based genetic information. The STRseqTyper122 kit genotypes 63 autosomal STRs, 16 X-STRs, 42 Y-STRs, and the Amelogenin locus. Amplicon sizes of 117 loci were below 300 bp. In this study, MiSeq FGx sequencing metrics for STRseqTyper122 were presented. The genotyping accuracy of this kit was examined by comparing to certified genotypes of NIST standard reference materials and results from five capillary electrophoresis-based kits. The sensitivity of STRseqTyper122 reached 125 pg, and > 80% of the loci were correctly called with 62.5 pg and 31.25 pg input genomic DNA. Repeatability, species specificity, and tolerance for DNA degradation and PCR inhibitors of this kit were also evaluated. STRseqTyper122 demonstrated reliable performance with routine case-work samples and provided a powerful tool for forensic applications. [ABSTRACT FROM AUTHOR]

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/μL in the minimum extraction volume (40 μL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R2, and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event. [ABSTRACT FROM AUTHOR]

Comparisons of DNA profiles recovered from the crime-scene and a person through short tandem repeat (STR) analysis using capillary electrophoresis (CE) is the gold standard method of determining a match. A comparison of STR profiles generated using CE to massively parallel sequencing (MPS) methods was undertaken. The results showed CE methods recovered high percentages of autosomal or Y-STR markers individually, however, MPS produced more information at low inputs and a higher number of STR markers overall, showing the potential of MPS to provide additional data to assist in solving major crimes. [ABSTRACT FROM AUTHOR]

We report on a comparison of the performance of the Applied Biosystems® SeqStudioTM Genetic Analyzer with the 3500 Genetic Analyzer when analysing trace DNA samples. Totally, 162 profiles generated from 'trace DNA' were analysed on both instruments with comparable number of alleles detected, relative fluorescence units (RFU) and likelihood ratio (LR) values. The SeqStudioTM was found to be suitable for the analysis of trace DNA samples. [ABSTRACT FROM AUTHOR]

The X chromosome is one of the two sex chromosomes found in humans and other mammals. It plays a crucial role in determining an individual's sex and contains genetic information that can be useful in forensic and human identity testing. Unlike autosomal DNA, which is inherited from both parents, the X chromosome is inherited differently in males and females, making it useful in certain types of analyses. In forensic investigations, the X chromosome can be used to determine the sex of an individual, which can be useful in identifying potential suspects or victims. Additionally, X chromosome analysis can be used to link evidence samples to a particular individual or to exclude individuals as potential contributors of the evidence. This can be particularly useful in cases where the evidence sample is a mixture of DNA from multiple individuals. In human identity testing, the X chromosome can be used in situations where other types of DNA analysis are not possible or inconclusive. For example, in cases where a potential parent is unavailable for testing, analysis of the X chromosome can be used to determine if a child is likely to be their biological offspring. Similarly, in cases where traditional autosomal DNA analysis is inconclusive, X chromosome analysis can be used to provide additional information about the biological relationship between individuals. However, there are some limitations to the use of X chromosome analysis in forensic and human identity testing. One limitation is that it is not as informative as autosomal DNA analysis, as it contains less genetic information. Additionally, the inheritance patterns of the X chromosome can be complex, particularly in cases where there are multiple generations involved. Therefore, X chromosome analysis should be interpreted in conjunction with other types of DNA analysis and other forms of evidence to ensure accurate and reliable results. Overall, the use of X chromosome analysis in forensic and human identity testing can provide important information in certain situations, particularly where traditional DNA analysis is not possible or inconclusive. As such, it is an important tool in the fields of forensic science and human identification. [ABSTRACT FROM AUTHOR]

There is interest in comparing the output, principally the likelihood ratio, from the two probabilistic genotyping software EuroForMix (EFM) and STRmix™. Many of these comparison studies are descriptive and make little or no effort to diagnose the cause of difference. There are fundamental differences between EFM and STRmix™ that are causative of the largest set of likelihood ratio differences. This set of differences is for false donors where there are many instances of LRs just above or below 1 for EFM that give much lower LRs in STRmix™. This is caused by the separate estimation of parameters such as allele height variance and mixture proportion using MLE under Hp and Ha for EFM. This can result in very different estimations of these parameters under Hp and Ha. It results in a departure from calibration for EFM in the region of LRs just above and below 1. [ABSTRACT FROM AUTHOR]

Perpetrators of offences missing from police files limit the capacity to investigate criminal behaviour for criminological research and operational purposes. Recent studies have shown that forensic DNA databanks, which include samples of DNA not yet matched to an individual, have the potential to address this problem. By examining information associated with criminal cases that involved DNA matches, we demonstrate that individuals who cannot be identified through DNA differ from those for whom such identification is possible. Based on data from 19 years of DNA matches in Quebec, Canada, we were able to assess the co-offending and repeat offending behavior of unidentified and identified individuals as well as the diversification, level of severity, and types of offenses. We found that the crimes of the 1,448 individuals who had not been identified were marginal as compared with those of individuals who had been identified. Unidentified individuals were more likely to act alone in repeated crimes, to be involved in fewer cases, to use less violence, and to become more specialized with increased activity. Our results are consistent with other studies that demonstrate that the criminal activity of unidentified individuals accords with the exposure hypothesis. The association of these findings with a network analysis approach is innovative and could have a greater than expected impact on investigations and policies, as well as having implications for forensic intelligence. [ABSTRACT FROM AUTHOR]

Generating degraded DNA samples can be useful for forensic DNA research as this can mimic the condition of crime scene samples and results from such samples. Methods which use quick and cost-effective ultraviolet light (UV) exposure can be utilized to degrade DNA but are not well documented. This study aimed to establish a procedure and define the parameters required to generate Short Tandem Repeat (STR) profiles at different degradation levels. UV exposure times between 0 and 60 s were tested using genomic and control DNA. Sample eluates were quantified with QuantifilerTM Trio pre- and post-UV exposure, and select samples were profiled using PowerPlex® 21. Quantification data and STR profiles were analysed to identify metrics which indicated degradation severity. Samples were observed to be degraded by UV exposure as evidenced by established DNA degradation markers. No specific metric was identified to reliably determine the severity of sample degradation, but it was noted that UV exposure between 5 and 10 s resulted in mild degradation, between 15 and 25 s generated moderate degradation, and between 45 and 60 s resulted in severe degradation in STR profiles. This study indicates that quantification before and after UV exposure may be useful to estimate the degree of degradation via one quantification target. [ABSTRACT FROM AUTHOR]

Although law enforcement use of commercial genetic genealogy databases has gained prominence since the arrest of the Golden State Killer in 2018, and it has been used in hundreds of cases in the United States and more recently in Europe and Australia, it does not have a standard nomenclature and scope. We analyzed the more common terms currently being used and propose a common nomenclature: investigative forensic genetic genealogy (iFGG). We define iFGG as the use by law enforcement of genetic genealogy combined with traditional genealogy to generate suspect investigational leads from forensic samples in criminal investigations. We describe iFGG as a proper subset of forensic genetic genealogy, that is, FGG as applied by law enforcement to criminal investigations; hence, investigative FGG or iFGG. We delineate its steps, compare and contrast it with other investigative techniques involving genetic evidence, and contextualize its use within criminal investigations. This characterization is a critical input to future studies regarding the legal status of iFGG and its implications on the right to genetic privacy.

Background: One thousand sixty-one individuals were sampled from the cities of Anbar, Baghdad, Basra, Diyala, Najaf, and Wasit in Iraq and typed for 15 forensic STRs to explore the genetic structure of Iraq and develop a forensic DNA database. The total number of alleles that were identified was 203. Result: Analyses of molecular variance (AMOVA) were then conducted Baghdad provides a good representation of the rest of the country, while Anbar is the most genetically distinct. The average heterozygosities of these loci was 0.779, homozygosities was 0.221, polymorphism information content was 0.77, power of discrimination was 0.927, and power of exclusion was 0.563. At these loci, a matching genotype will occur, on average, in 1 in 8.152 × 1017 individuals. For paternity tests, the average paternity probability for a matching profile is 99.9997%. Conclusions: These loci are appropriate for use in forensic and paternity testing for this population. Iraq is similar to other countries in the Middle East, particularly Iran and Turkey, and is more similar to Europe than either Asia or Africa. [ABSTRACT FROM AUTHOR]

Autosomal short tandem repeat (STR) population data collected from a well characterized population are needed to correctly assigning the weight of DNA profiles in the courtroom and widely used for ancestral analyses. In this study, allele frequencies for the 15 autosomal short tandem repeat (STR) loci included in the AmpFlSTR® Identifiler® plus kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818, FGA) were obtained by genotyping 332 unrelated individuals of Ghanaian origin. Statistical tests on STR genotype data showed no significant departure from Hardy–Weinberg equilibrium (HWE). The overall match probability, combined power of exclusion and combined power of discrimination for these loci were 1 in 3.85 × 1017, 0.99999893 and 0.99999998, respectively. Polymorphic information content (PIC) greater than 0.70 was observed for all loci except TH01 and D13S317. These statistical parameters confirm that this combination of loci is valuable for forensic identification and parentage analysis. Our results were also compared with those for 20 other human populations analyzed for the same set of markers. We observed that the Ghanaian population grouped with other African populations in two-dimensional principal coordinate (PCO) and a neighbor-joining (N-J) data mapping and placed closest to Nigerians. This observation reflects cultural similarities and geographical factors, coupled with the long history of migration and trading activities between Ghana and Nigeria. Our report provides what we believe to be the first published autosomal STR data for the general Ghanaian population using 15 loci genotyped using the AmpFlSTR® Identifiler® plus kit methodology. Our data show that the loci tested have sufficient power to be used reliably for DNA profiling in forensic casework and help to elucidate the genetic history of people living in the country. [ABSTRACT FROM AUTHOR]

• Contact with liquid saliva effectively transfers foreign DNA beneath fingernails. • When dried, DNA from saliva does not readily transfer beneath fingernails. • Wet saliva is effectively removed from beneath fingernails by hand-washing. • Substantial decreases in salivary DNA are observed 24 h following deposition. This study investigated the transfer and persistence of salivary DNA under fingernails. This was performed to address a common alternate hypothesis presented to scientists in court, asserting that a relatively large quantity of DNA detected beneath the fingernails, typically from a victim of crime, originates from innocuous transfer of saliva in a casual setting. It was determined through these studies that contact with liquid saliva was an effective way to transfer foreign DNA beneath fingernails. However, when saliva was dried, DNA did not readily transfer through casual contact. When liquid saliva was placed directly beneath fingernails the amount of DNA detected from the saliva donor twenty-four hours later was several hundred-fold lower than the amount detected when sampling occurred immediately following deposition. Furthermore, when the recipients' hands were washed immediately following the deposition of liquid saliva beneath fingernails, the majority of foreign DNA was removed following one hand washing and all detectable foreign DNA was removed from most recipients' hands after three or six hand washings. This study demonstrates that casual contact with wet saliva can result in the transfer of substantial quantities of DNA beneath fingernails but that it does not typically persist for extended periods of time and is mostly removed if the hands are washed soon after deposition. [ABSTRACT FROM AUTHOR]

This study assessed the usefulness of DNA quantification to predict the success of historical samples when analyzing SNPs, mtDNA, and STR targets. Thirty burials from six historical contexts were utilized, ranging in age from 80 to 800 years postmortem. Samples underwent library preparation and hybridization capture with two bait panels (FORCE and mitogenome), and STR typing (autosomal STR and Y-STR). All 30 samples generated small (~80 bp) autosomal DNA target qPCR results, despite mean mappable fragments ranging from 55–125 bp. The qPCR results were positively correlated with DNA profiling success. Samples with human DNA inputs as low as 100 pg resulted in ≥80% FORCE SNPs at 10X coverage. All 30 samples resulted in mitogenome coverage ≥100X despite low human DNA input (as low as 1 pg). With PowerPlex Fusion, ≥30 pg human DNA input resulted in >40% of auSTR loci. At least 59% of Y-STR loci were recovered with Y-target qPCR-based inputs of ≥24 pg. The results also indicate that human DNA quantity is a better predictor of success than the ratio of human to exogenous DNA. Accurate quantification with qPCR is feasible for historical bone samples, allowing for the screening of extracts to predict the success of DNA profiling. [ABSTRACT FROM AUTHOR]

Objective: To assess the pre-analytical factors including hand-side, representative collection technique and individual forensic physician for foreign deoxyribonucleic acid (DNA) detection in specimen collected from deceased’s fingernails. Material and Methods: This cross-sectional study, of an initial 164 samples from the fingernails of both hands, of 82 deceased caseworks; from 2010 to 2018, at the Forensic Unit, Faculty of Medicine, Prince of Songkla University. The autosomal short tandem repeats profile fingernail DNA results, pre-analytical factors, and the deceased’s characteristics were obtained from the records. The fingernail DNA outcomes were evaluated and ranked into five groups, i.e., high-level profile, low-level profile, or residual profile; if foreign alleles of more than 11 alleles, 4 to 11 alleles, or less than 4 alleles were detected, respectively. The non-specific profile group consisted of foreign DNA being detected; however the peak signals were below the decisional threshold. The unidentified group consisted of no foreign DNA being detected. The full model underwent both directional stepwise model selection, and the resulting model with the lowest Akaike information criterion was selected as the final model. The final model was analyzed by ordinal logistic regression for significant associated factors: at a 95% confidence level. Results: The representative collection technique is an associated factor, via the use of fingernail swabs (adjusted odds ratio (ORa)=13.44, 95% confidence interval (CI)=2.89–62.45), and had a larger effect size than using fingernail cuttings (ORa=6.84, 95% CI=1.47–31.86). Conclusion: At post-mortem examination, for the collection of foreign DNA from fingernails, the use of fingernail swabs, as a collection technique, is of particular interest.

The validity of a probabilistic genotyping (PG) system is typically demonstrated by following international guidelines for the developmental and internal validation of PG software. These guidelines mainly focus on discriminatory power. Very few studies have reported with metrics that depend on calibration of likelihood ratio (LR) systems. In this study, discriminatory power as well as various calibration metrics, such as Empirical Cross-Entropy (ECE) plots, pool adjacent violator (PAV) plots, log likelihood ratio cost (Cllr and Cllr cal ), fiducial calibration discrepancy plots, and Turing' expectation were examined using the publicly-available PROVEDIt dataset. The aim was to gain deeper insight into the performance of a variety of PG software in the 'lower' LR ranges (∼LR 1–10,000), with focus on DNAStatistX and EuroForMix which use maximum likelihood estimation (MLE). This may be a driving force for the end users to reconsider current LR thresholds for reporting. In previous studies, overstated 'low' LRs were observed for these PG software. However, applying (arbitrarily) high LR thresholds for reporting wastes relevant evidential value. This study demonstrates, based on calibration performance, that previously reported LR thresholds can be lowered or even discarded. Considering LRs >1, there was no evidence for miscalibration performance above LR ∼1000 when using Fst 0.01. Below this LR value, miscalibration was observed. Calibration performance generally improved with the use of Fst 0.03, but the extent of this was dependent on the dataset: results ranged from miscalibration up to LR ∼100 to no evidence of miscalibration alike PG software using different methods to model peak height, HMC and STRmix. This study demonstrates that practitioners using MLE-based models should be careful when low LR ranges are reported, though applying arbitrarily high LR thresholds is discouraged. This study also highlights various calibration metrics that are useful in understanding the performance of a PG system. • Discriminatory power and calibration performance of PG software are evaluated. • The utility of various calibration metrics are explored in 'low' LR ranges. • Focus was on DNAStatistX and EuroForMix software using the MLE method. • Calibration performance was dependent on Fst value and dataset size. • Results suggest reconsideration of lower LR thresholds and cautious reporting of 'low' LRs. [ABSTRACT FROM AUTHOR]

Aim The present study was designed to explore the STR diversity and genomic history of the inhabitants of Nayagarh district of Odisha, India. We also tested the proficiency of the most recent, new generation PowerPlexR Y23 multiplex system for forensic characterisation and to decipher the phylogenetic affinities. Subjects and methods The genetic diversity and polymorphism among 236 healthy unrelated male volunteers from Nayagarh district of Odisha, India was investigated. This investigation was carried out via 23 Y-chromosomal STRs using capillary electrophoresis. Result A total 223 unique haplotypes were reported. Discrimination capacity (DC), gene diversity (GD) and power of discrimination (PD) were observed as 0.945, 0.999999999998333, and 0.99999999999794, respectively. Polymorphic information content (PIC) and matching probability (PM) were reported as 0.999999999925535 and 2.06 × 10−12, respectively. Simultaneously, the haplogroup analysis characterised with C2, E1b1a, E1b1b, G2a, H1, I2a, J2a, J2b, L, O, O1, O2, Q, R1a, R2, and T haplogroups, disclosing the possible geographical relatedness of the studied population to different areas of the world. Conclusion Phylogenetic analysis with previously reported Indian and Asian populations showed the genetic closeness of the studied population to different Indian populations and the Bangladeshi population of Dhaka, whereas the Bhotra population of Odisha and Han population of China showed much less genetic affinity.

This review paper covers the forensic-relevant literature in biological sciences from 2019 to 2022 as a part of the 20th INTERPOL International Forensic Science Managers Symposium. Topics reviewed include rapid DNA testing, using law enforcement DNA databases plus investigative genetic genealogy DNA databases along with privacy/ethical issues, forensic biology and body fluid identification, DNA extraction and typing methods, mixture interpretation involving probabilistic genotyping software (PGS), DNA transfer and activity-level evaluations, next-generation sequencing (NGS), DNA phenotyping, lineage markers (Y-chromosome, mitochondrial DNA, X-chromosome), new markers and approaches (microhaplotypes, proteomics, and microbial DNA), kinship analysis and human identification with disaster victim identification (DVI), and non-human DNA testing including wildlife forensics. Available books and review articles are summarized as well as 70 guidance documents to assist in quality control that were published in the past three years by various groups within the United States and around the world.

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Previous studies examining whether splitting the DNA extract for replicate amplification versus maximizing the template available for a 'one-shot' amplification either examined the benefits of using replicates (without a comparison to a single amplification), or used semi-continuous probabilistic software that ignores peak height information. In this study, we use a fully continuous probabilistic genotyping software to compare the effectiveness of amplifying a single sample compared to splitting the sample and conducting a joint analysis of replicate amplifications. We show that the one-shot approach is marginally better than splitting the DNA extract across a range of contributor numbers and template amounts. Where there is unexpected peak height variability or drop-in within the profile not modelled during interpretation, a replicate approach may be better. [ABSTRACT FROM AUTHOR]

A new calculation module within the PopStats module of the CODIS software package, based on the underlying mathematics presented in the MixKin software package, has been developed for assigning the Likelihood Ratio (LR) of DNA mixture profiles. This module uses a semi-continuous model that allows for population structure and allelic drop-out and drop-in but does not require allelic peak heights or other laboratory-specific parameters. This new implementation (named SC Mixture), like MixKin, does not specify or estimate a probability of drop-out. Instead, each contributor to a mixture has an independent drop-out rate, and the probability of the mixture profile for a specified proposition concerning the contributors is integrated over the range of possible drop-out rates. The allelic drop-in rate and the population structure parameter, theta, used by the software are specified by the user. The user can examine up to five contributors to a mixture, however, conditioning on assumed contributors and limiting the number of unknowns in both numerator and denominator hypotheses greatly improves performance. We report results from an extensive validation study performed for ten mixtures with each of one (single source), two, three, four, or five contributors, with four combinations of drop-in rate and a population structure parameter. Each mixture was run as a complete profile or with the random removal of alleles to simulate drop-out. All 1620 combinations were evaluated with PopStats, MixKin, and LRmix and considerable consistency was found among the results with all three packages.

Crime laboratories have been faced with large casework backlogs due to lengthy processing times, limited resources and scientists, and rising crime rates. Evidence related to sexual assault crimes, specifically sexual assault kits (SAKs), heavily contribute to the reported backlogs. Although more sensitive, faster chemistries and automated techniques have been implemented over the years, the traditional STR workflow remains relatively unchanged. Enhanced workflows such as direct PCR and Rapid DNA have the potential to streamline the processing of forensic evidence items including those commonly submitted in SAKs, but the FBI QAS guidelines restrict CODIS‐approved labs from implementing these methods for forensic samples. Recent studies have shown decreased turnaround times and improved or comparable profiling success with both approaches. However, review of the literature shows a lack of in‐depth research comparing traditional DNA workflows to faster and more sensitive direct PCR and/or Rapid DNA approaches for evidentiary samples, especially for SAKs. By providing the forensic science and criminal justice communities with the strengths and limitations of direct PCR and Rapid DNA methods, stakeholders and policy makers may be better informed. [ABSTRACT FROM AUTHOR]

DNA analyses from challenging samples such as touch evidence, hairs and skeletal remains push the limits of the current forensic DNA typing technologies. Reverse complement PCR (RC-PCR) is a novel, single-step PCR target enrichment method adapted to amplify degraded DNA. The sample preparation process involves a limited number of steps, decreasing the labor required for library preparation and reducing the possibility of contamination due to less sample manipulation. These features of the RC-PCR make the technology a unique application to successfully target single nucleotide polymorphisms (SNPs) in fragmented and low copy number DNA and yield results from samples in which no or limited data are obtained with standard DNA typing methods. The developed RC-PCR short amplicon 85 SNP-plex panel is a substantial improvement over the previously reported 27-plex RC-PCR multiplex that will provide higher discrimination power for challenging DNA sample analyses.

Every year thousands of people disappear in Brazil. However, it is still not clear how many of these are related to cases of forced disappearance resulting from institutional and state violence. A growing demand arising from family members of missing persons has resulted in creating of the Human Identification Lab at the Center of Anthropology and Forensic Archeology at the Federal University of São Paulo, São Paulo, Brazil. The intersection between the struggles of family members and the forensic knowledge at the university enabled the development of methodologies and protocols divided into three stages: ante mortem; post mortem, and forensic genetic, specifically built to identify victims of forced disappearance.

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g. , in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1–15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures. • Use of UMIs reduced sequencing errors more than tenfold. • UMIs lead to drastically lowered stutter ratios, from 9.5% ± 5.3% to 2.1% ± 1.3%. • The method generated complete single-source profiles for 62.5 pg DNA. • Minor contributors down to 1% were detected in mixtures. [ABSTRACT FROM AUTHOR]

CRISPR-Cas technology has revolutionized the field of biotechnology with its precise therapeutic use from genetic as well as infectious diseases point of view. This technology is rapidly evolving to single tool enabling site-directed cut in the genome and highly specific activation or inhibition of gene expression or the exchange of single bases. Besides clinical applications, CRISPR-Cas technology has also shown promising use in the field of forensic DNA analysis. Enrichment of targeted genetic marker for identification followed by sequencing and non-PCR–dependent technique ensures the use of CRISPR-Cas technology in challenging forensic biological samples. The use of this advanced technology is also deemed helpful in mixed profile attribution, mostly in LCN contributors and the generation of a useful DNA profile in degraded samples. Besides its useful applications in forensic DNA analysis, CRISPR-Cas technology poses a huge threat from the generation of ghost DNA profiles by modification/alteration of target genetic markers. Forensic DNA analysts should carry out analysis of additional markers such as non-CODIS markers, Y-, X-chromosome markers, and mitochondrial DNA sequencing in a suspected ghost DNA profile case. Key points: • CRISPR-Cas9 technique is useful in analyzing LCN, mixed and degraded samples • Alteration of DNA using this technique can lead to generation of ghost DNA profiles • Alternative genetic markers and methylation pattern may detect a ghost DNA profile [ABSTRACT FROM AUTHOR]

The extremely high nature of transmissibility and severity of infection due to the novel Corona virus is a serious threat to mankind. This letter delivers a caution for forensic DNA experts in the era of COVID-19 infection from the Indian perspective. Samples are routinely transported to laboratories without any specific guidelines. Therefore, this is high time to formulate clear guidelines for the handling of biological material, from receiving to processing in the laboratory during such a pandemic

Interpreting forensic DNA signal is arduous since the total intensity is a cacophony of signal from noise, artifact, and allele from an unknown number of contributors (NOC). An alternate to traditional bulk‐processing pipelines is a single‐cell one, where the sample is collected, and each cell is sequestered resulting in n single‐source, single‐cell EPGs (scEPG) that must be interpreted using applicable strategies. As with all forensic DNA interpretation strategies, high quality electropherograms are required; thus, to enhance the credibility of single‐cell forensics, it is necessary to produce an efficient direct‐to‐PCR treatment that is compatible with prevailing downstream laboratory processes. We incorporated the semi‐automated micro‐fluidic DEPArray™ technology into the single‐cell laboratory and optimized its implementation by testing the effects of four laboratory treatments on single‐cell profiles. We focused on testing effects of phosphate buffer saline (PBS) since it is an important reagent that mitigates cell rupture but is also a PCR inhibitor. Specifically, we explored the effect of decreasing PBS concentrations on five electropherogram‐quality metrics from 241 leukocytes: profile drop‐out, allele drop‐out, allele peak heights, peak height ratios, and scEPG sloping. In an effort to improve reagent use, we also assessed two concentrations of proteinase K. The results indicate that decreasing PBS concentrations to 0.5X or 0.25X improves scEPG quality, while modest modifications to proteinase K concentrations did not significantly impact it. We, therefore, conclude that a lower than recommended proteinase K concentration coupled with a lower than recommended PBS concentration results in enhanced scEPGs within the semi‐automated single‐cell pipeline. [ABSTRACT FROM AUTHOR]

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. The use of the optimised workflow on aged archived latent fingerprint (ALFP) lift card samples (aged 2–28 years) improves the number of STR alleles recovered, providing more discriminatory STR profiles than those processed using the traditional workflow. Interpretable STR alleles can be detected from ALFP lift card samples stored as long as 28 years when the optimised procedures are followed. The use of individual laboratory-sterilised tools for sample preparation and the addition of a re-purification step with Centri-Sep columns in the recommended optimised workflow seem to limit the ability to detect low-level secondary DNA sources. [ABSTRACT FROM AUTHOR]

The present study was designed to explore the STR diversity and genomic history of the inhabitants of Nayagarh district of Odisha, India. We also tested the proficiency of the most recent, new generation PowerPlexR Y23 multiplex system for forensic characterisation and to decipher the phylogenetic affinities. The genetic diversity and polymorphism among 236 healthy unrelated male volunteers from Nayagarh district of Odisha, India was investigated. This investigation was carried out via 23 Y-chromosomal STRs using capillary electrophoresis. A total 223 unique haplotypes were reported. Discrimination capacity (DC), gene diversity (GD) and power of discrimination (PD) were observed as 0.945, 0.999999999998333, and 0.99999999999794, respectively. Polymorphic information content (PIC) and matching probability (PM) were reported as 0.999999999925535 and 2.06 × 10−12, respectively. Simultaneously, the haplogroup analysis characterised with C2, E1b1a, E1b1b, G2a, H1, I2a, J2a, J2b, L, O, O1, O2, Q, R1a, R2, and T haplogroups, disclosing the possible geographical relatedness of the studied population to different areas of the world. Phylogenetic analysis with previously reported Indian and Asian populations showed the genetic closeness of the studied population to different Indian populations and the Bangladeshi population of Dhaka, whereas the Bhotra population of Odisha and Han population of China showed much less genetic affinity. [ABSTRACT FROM AUTHOR]

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.Key points The use of the optimised workflow on aged archived latent fingerprint (ALFP) lift card samples (aged 2–28 years) improves the number of STR alleles recovered, providing more discriminatory STR profiles than those processed using the traditional workflow. Interpretable STR alleles can be detected from ALFP lift card samples stored as long as 28 years when the optimised procedures are followed. The use of individual laboratory-sterilised tools for sample preparation and the addition of a re-purification step with Centri-Sep columns in the recommended optimised workflow seem to limit the ability to detect low-level secondary DNA sources.

As part a body donation program, blood samples were collected and stored on untreated (non-FTA) blood cards. The blood cards were evaluated in terms of DNA preservation and STR typing success with resulting profiles assessed with special consideration given to profile matching for positive identification and biogeographic ancestry estimation. While STR profiles were successfully generated for all samples, results indicate that the time interval between date of death and sample collection have an impact on DNA quantity and quality. There is a statistically significant decrease in relative fluorescent unit (RFU) values with increasing time interval between date of death and sample collection, indicating degradation in the blood card samples related to the post-mortem interval prior to sample collection. The STR profiles were used to estimate ancestry and admixture using the program STRUCTURE, demonstrating utility of these markers beyond individual identification purposes, with caveats for application based on population history.

The evaluation of forensic DNA expert opinions (in some countries expert witness testimonies) and the way it affects criminal judgement is of paramount importance. We have selected one of the largest challenges when it comes to the evaluation of forensic DNA evidence, contamination of DNA samples, and examined how it influences the decisions judges make about the credibility of DNA evidence in Hungary. [ABSTRACT FROM AUTHOR]

We analyzed the accumulation of population polymorphism in 2504 individuals - nuclear genomes (nDNA) of 26 populations (81 genes associated to extreme environments) and 3295 mitochondrial genomes (mtDNA) of 47 populations with the aim to found mitonuclear relationship associated an extremes environment as altitude. For that, we use an algorithm developed by us to determine the accumulation of polymorphisms by segments in the genome and thus be able to perform the multivariate analysis to found SNPs differences and similarities among populations. The results showed in Peruvian population a statistically significant mitonuclear relationship for 113/293970 nDNA SNPs in 16/81 genes. In the case of the mtDNA, we found a statistically significant mitonuclear relationship for 6/22 mtDNA positions – Gene. Additionally for the Peruvian population, the MRPP3 had the greatest polymorphism contribution with respect to other populations. Then, these nDNA and mtDNA SNPs in genetically close populations to Peru can be applied to forensic genomic phenotyping to identify groups likely adapted to extreme conditions (such as altitude) or make individualization between low and high altitude populations. [ABSTRACT FROM AUTHOR]

BackgroundTechnological advances have enabled the analysis of very small amounts of DNA in forensic cases. However, the DNA profiles from such evidence are frequently incomplete and can contain contributions from multiple individuals. The complexity of such samples confounds the assessment of the statistical weight of such evidence. One approach to account for this uncertainty is to use a likelihood ratio framework to compare the probability of the evidence profile under different scenarios. While researchers favor the likelihood ratio framework, few open-source software solutions with a graphical user interface implementing these calculations are available for practicing forensic scientists.ResultsTo address this need, we developed Lab Retriever, an open-source, freely available program that forensic scientists can use to calculate likelihood ratios for complex DNA profiles. Lab Retriever adds a graphical user interface, written primarily in JavaScript, on top of a C++ implementation of the previously published R code of Balding. We redesigned parts of the original Balding algorithm to improve computational speed. In addition to incorporating a probability of allelic drop-out and other critical parameters, Lab Retriever computes likelihood ratios for hypotheses that can include up to four unknown contributors to a mixed sample. These computations are completed nearly instantaneously on a modern PC or Mac computer.ConclusionsLab Retriever provides a practical software solution to forensic scientists who wish to assess the statistical weight of evidence for complex DNA profiles. Executable versions of the program are freely available for Mac OSX and Windows operating systems.

The developmental validation of the software DBLR™ (‘database likelihood ratios’) is described. DBLR™ implements a flexible framework for the assignment of likelihood ratios for forensic DNA profiles. This framework allows the user to assign a likelihood ratio for virtually any conceivable scenario using any combination of single-source or mixed DNA profiles. Propositions are defined by constructing pedigrees and then linking DNA typing results to pedigree individuals. Complex kinship scenarios including incestuous relationships can be evaluated. Additional functionality within DBLR™ allows the user to explore the discriminatory power of any DNA profile (Explore Deconvolution), search a profile against a database of known profiles using flexible search strategies (Search Database), investigate whether two mixtures share one or more common donors (Mixture to Mixture), and search a database of known profiles for an assumed common donor between two or more profiles (Common Donor). Developmental validation was carried out in accordance to the FBI Quality Assurance Standards for Forensic DNA Testing Laboratories (2020) and included functional and reliability testing as well as accuracy, precision, sensitivity, and specificity studies. The results of developmental validation demonstrate that each of the functions within DBLR™ is performing as expected.

In forensic DNA casework software tools aid in the data management and efficient, with low risk for error, interpretation and comparison of DNA profiling data. The need for such software has grown with increased numbers of genetic markers, more sensitive analysis methods and growing numbers of crime scene samples send for DNA analysis. To that end, the DNA eXpert System, DNAxs, was developed. This study describes how the expert system was realized and how it evolved during the first three years after the initial implementation. Insight is given in the software architecture, its modular design enabling to communicate with various software systems and how this system was implemented in a casework setting. The importance of quality aspects are highlighted, such as (automated) software testing at various levels. The code coverage is presented as well as the numbers of software bugs that were discovered. These bugs were more severe in earlier software versions than in the more recent versions and did not affect the interpretation of DNA results. The usefulness of the overall software suite and automated steps in DNA profile interpretation were evaluated based on its usage in forensic DNA casework during the first three years after its implementation at the developing laboratory. Because of automated profile comparisons, cases with larger numbers of profiles can be handled, are less prone to error and are extremely less time consuming. The implementation of an integrated quantitative probabilistic genotyping module into DNAxs enabled a more efficient workflow as no longer data files have to be exported and imported into different software. Extensive automated software tests and an audit trail serve as quality aspects for the usage of DNAxs. In times of the pandemic this software was found even more valuable than ever thought as it enabled working from home and it proved robust when used with many simultaneous users. The DNAxs software is regarded future proof and many new features and applications are envisioned.

Y-chromosome short tandem repeat (Y-STR) markers have been widely used in forensic applications and usually show monoallelic or diallelic genotypic patterns at certain double-copied loci. In this study, we have found 13 samples among 703 males with multi-alleles at the DYS385ab locus, including one with five mutant alleles, nine with four, and three with three. The frequency of abnormal DYS385ab genotypes was 1.85% (13/703), which is very high in the Han Chinese population. The percentage of samples with diallelic patterns at DYS385ab was higher than that of monoallelic patterns (80.23% vs. 17.92%). Additionally, the percentage of samples with tetra-allelic patterns at DYS385ab was higher than that of tri-allelic patterns (1.28% vs. 0.43%), suggesting that there are possibly two copies with duplicated events happening frequently on the Y chromosome. Interestingly, the peak height of allele 13 was two to three-folds higher than that of other alleles. The allele 18 peak height was also two-fold higher than others, which could potentially be explained by a duplication event mechanism. We also found that tri-allelic genotypes for alleles 13, 17, and 20, tetra-allelic genotypes for alleles 13, 14, 19, and 20, and tetra-allelic genotypes for alleles 12, 13, 19 and 21 were more common than others. Furthermore, all 13 samples had multi-alleles containing allele 13, implying a founder effect in this particular Chinese-specific ethnic group. Taken together, this study provides new information for this population and will be useful for paternal lineage identification, kinship analysis, and family relationship reconstruction using Y-STR forensic DNA analysis methods. [ABSTRACT FROM AUTHOR]

An increasing number of US jurisdictions have begun to submit their previously untested sexual assault kits (SAKs) for DNA testing. However, best practices for what should happen after testing are not well established. Should all cases be investigated regardless of the testing outcome or only those that returned a DNA hit? We examine an early‐adopter jurisdiction that has completed testing and investigating all 5165 previously never tested kits. We explore and compare the criminal justice outcomes and cost‐effectiveness of investigating: all cases, those with CODIS hits, and those without CODIS hits. Findings indicate the SAK initiative produced a cost savings to the community: $26.48 million ($5127 p/kit) after the inclusion of tangible and intangible costs of future sexual assaults averted through convictions, of which $9.99 million ($1934 p/kit) was from also investigating no CODIS hit cases. When considering only the costs to law enforcement, investigating all cases cost $12,000 p/additional conviction. Findings also illustrate the cost‐effectiveness of investigating no CODIS hits cases and support an "investigate all" approach. This study enhances our understanding of the economic value of what comes after testing kits and investigating cases and provides a framework for jurisdictions for prioritizing resources and maximizing outcomes from testing. [ABSTRACT FROM AUTHOR]