It's all relative: A multi-generational study using ForenSeq™ Kintelligence.

The successful application of Forensic Investigative Genetic Genealogy (FIGG) to the identification of unidentified human remains and perpetrators of serious crime has led to a growing interest in its use internationally, including Australia. Routinely, FIGG has relied on the generation of high-density single nucleotide polymorphism (SNP) profiles from forensic samples using whole genome array (WGA) (∼650,000 or more SNPs) or whole genome sequencing (WGS) (millions of SNPs) for DNA segment-based comparisons in commercially available genealogy databases. To date, this approach has required DNA of a quality and quantity that is often not compatible with forensic samples. Furthermore, it requires the management of large data sets that include SNPs of medical relevance. The ForenSeq™ Kintelligence kit, comprising of 10,230 SNPs including 9867 for kinship association, was designed to overcome these challenges using a targeted amplicon sequencing-based method developed for low DNA inputs, inhibited and/or degraded forensic samples. To assess the ability of the ForenSeq™ Kintelligence workflow to correctly predict biological relationships, a comparative study comprising of 12 individuals from a family (with varying degrees of relatedness from 1st to 6th degree relatives) was undertaken using ForenSeq™ Kintelligence and a WGA approach using the Illumina Global Screening Array-24 version 3.0 Beadchip. All expected 1st, 2nd, 3rd, 4th and 5th degree relationships were correctly predicted using ForenSeq™ Kintelligence, while the expected 6th degree relationships were not detected. Given the (often) limited availability of forensic samples, findings from this study will assist Australian Law enforcement and other agencies considering the use of FIGG, to determine if the ForenSeq™ Kintelligence is suitable for existing workflows and casework sample types considered for FIGG.

Discordance of Penta D x.4 microvariant alleles results between three capillary electrophoresis and one massively parallel sequencing short tandem repeat kits.

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5' flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3' region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings.

'Low' LRs obtained from DNA mixtures: On calibration and discrimination performance of probabilistic genotyping software.

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 Cllrcal), 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.

Does Sunlight Affect the Quality for Purposes of DNA Analysis of Blood Stain Evidence Collected from Different Surfaces?

The aim of this study was to investigate the effect of sunlight on the degradation of DNA samples taken from blood stains from different types of surfaces. A blood sample obtained from a single male donor was placed on seven different surfaces (galvanized sheet, iron rod, newspaper, white printer paper, glass, soil, and ceramic panel). Samples were kept, during a 4-week summer period, in a room, but next to an open window. Every 7 days, 1 mm2 of blood sample was collected from each substrate and stored in labeled tube for later analysis. DNA was extracted with the Chelex method, amplified using AmpFISTRTM MinifilerTM Plus Amplification Kit, and quantified using a QuantifilerTM Human DNA Quantification kit. After 7 days of sun exposure, the highest DNA concentration was determined to be from the sample from a galvanized sheet stain, followed by, in order of decreasing concentration, the ceramic panel, glass, newspaper, iron rod, and white printer paper surface. As expected, the DNA concentration from all samples decreased as the sunlight exposure time progressed. The results obtained after the amplification in the MiniFilerTM system were in correlation with the DNA concentrations measured by the qPCR method for all samples, except for the glass, soil, and white printer paper samples. The obtained data show that DNA degradation is correlated to the length of sunlight exposure and to the type of surface the samples are collected from. A negative qPCR result does not mean negative PCR amplification in the STR system; therefore, both methods should be applied when analyzing forensic samples collected from trace evidence.

A systematic approach to the analysis of illicit drugs for DNA with an overview of the problems encountered.

Due to the restricted nature of illicit drugs, it is difficult to conduct research surrounding the analysis of this drug material for any potential DNA in sufficient quantities acceptable for high numbers of replicates. Therefore, the current research available in peer reviewed journals thus far regarding analysing illicit drugs for DNA has been performed under varying experimental conditions, often using surrogate chemicals in place of illicit drugs. The data presented within this study originated from the analysis of genuine illicit drugs prepared both in controlled environments and those seized at the Australian border (and therefore from an uncontrolled environment) to determine if DNA can be obtained from this type of material. This study has been separated into three main parts (total n=114 samples): firstly, methamphetamine synthesised within a controlled environment was spiked with both saliva and trace DNA to determine the yield following DNA extraction; secondly, methamphetamine also synthesised in a controlled environment but on a larger scale was tested for the amount of DNA added incidentally throughout the synthesis, including the additional steps of recrystallising, homogenising and "cutting" the drug material to simulate preparation for distribution; and thirdly, the detection of human DNA within samples of cocaine and heroin seized at the Australian border. The DNA Fast Flow Microcon Device was utilised to concentrate all replicates from the same source into one combined extract to improve the DNA profiles for the samples where no DNA spiking occurred. Full STR profiles were successfully obtained from drug samples spiked with both saliva and trace DNA. Methamphetamine was present in the final DNA extracts and caused incompatibilities with the quantification of DNA using Qubit. The yields of DNA from drugs not spiked with DNA sources were much lower, resulting in 36 % of samples yielding alleles where all others did not. These results were not unexpected given these were realistic drug samples where the history of the drug material was unknown. This is the first study to obtain DNA profiles from genuine illicit drug material in both controlled and uncontrolled environments and indicates that the analysis of illicit drugs for DNA is an avenue worth pursuing to provide information which can in turn assist with disrupting the supply of these drugs. Given that DNA profiling is carried out worldwide using essentially the same systems as described within this study, the potential for impact is on a national and international scale.

Modified differential lysis for sexual assault samples using a combined enzymatic and alkaline approach.

Sexual assault sample processing, despite recent funding and research efforts, remains time-consuming, labourious, and inefficient. These limitations, combined with the prevalence of sexual assaults, have prompted the need to develop a cheaper, quicker, and more robust method for separating victim and perpetrator contributions within sexual assault evidence so that analysts can keep pace with submissions and cases can be resolved in a timely manner. Thus, this study examined the use of a combined enzymatic and alkaline approach for differential cell lysis-with the goal of developing a quick, cheap, and more efficient DNA isolation method. Quantification results for this assay revealed that (72.0 ± 18.3)%, (15.8 ± 14.2)%, and (29.5 ± 23.7)% of total DNA were retained in sperm fractions for neat semen, neat vaginal, and semen-vaginal mixture eluates, respectively. Short tandem repeat (STR) analysis of mixture samples processed with this technique exhibited sperm fraction DNA profiles with mean male-to-female ratios of 1.74:1, which was a 3.01 ± 2.30-fold improvement in male-to-female ratios and led to the recovery of 5.90 ± 7.80 unshared male contributor alleles in sperm fractions that were otherwise undetected in unseparated controls. Overall, this study presented a modified differential lysis approach using prepGEM™ and sodium hydroxide treatments that can accomplish cell elution and fractional lysis within 25 min. Future studies should investigate alternative "non-sperm" cell lysis methods to enhance lysis efficiency and minimize the potential for inhibition, as well as the optimization and automation of this technique. Key points: Traditional sexual assault sample processing methods are time-consuming and inefficient.This modified differential lysis method produces lysates with sufficient DNA yield and quality.A combined technique using enzymatic and alkaline lysis can accomplish fractional separation.Lysis with prepGEM and NaOH absent purification is compatible with downstream processes.

Looking into the Quantification of Forensic Samples with Real-Time PCR.

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.

Extending the discussion on inconsistency in forensic decisions and results.

The subject of inter- and intra-laboratory inconsistency was recently raised in a commentary by Itiel Dror. We re-visit an inter-laboratory trial, with which some of the authors of this current discussion were associated, to diagnose the causes of any differences in the likelihood ratios (LRs) assigned using probabilistic genotyping software. Some of the variation was due to different decisions that would be made on a case-by-case basis, some due to laboratory policy and would hence differ between laboratories, and the final and smallest part was the run-to-run difference caused by the Monte Carlo aspect of the software used. However, the net variation in LRs was considerable. We believe that most laboratories will self-diagnose the cause of their difference from the majority answer and in some, but not all instances will take corrective action. An inter-laboratory exercise consisting of raw data files for relatively straightforward mixtures, such as two mixtures of three or four persons, would allow laboratories to calibrate their procedures and findings.

Opt-in or out? Public perspectives on forensic DNA kinship investigations within the Dutch-speaking community.

Forensic DNA kinship investigation involves analyzing genetic relationships between individuals to offer new leads for solving (cold) cases. Familial DNA matching has become a valuable asset in criminal case investigations, especially when traditional DNA methods hit dead ends. However, concerns surrounding ethical and privacy implications raised questions about its implementation and acceptance among the general public. The present study investigated the public perspectives regarding forensic DNA kinship investigations among 1710 Dutch-speaking Belgians using an online cross-sectional survey. The questionnaire consisted of three categories, including personal information, DNA knowledge, and their opinion on several familial DNA searching and investigative genetic genealogy related questions. The participants' average DNA knowledge score was 71 %, indicating a relatively high level of understanding of DNA-related concepts. Remarkably, the study revealed that 92 % of the participants expressed willingness to cooperate as a volunteer in a forensic DNA kinship investigation, irrespective of their scientific background or educational level. Key factors influencing participation included assurance of painless sampling and robust privacy safeguards. Participants lacking familiarity with DNA hesitated more towards participating in forensic DNA analysis, referring to "the fear of the unknown". Despite ethical and privacy concerns, the highly positive attitude towards forensic DNA analysis reflects a level of empathy and willingness to contribute to the pursuit of justice. Nearly all participants (95 %) agreed to use online DNA databases for resolving violent crimes with forensic genetic genealogy, but half emphasized the need for prior informed consent, referring to the current "opt-in" system. The results underscore the need for stringent regulations and ethical oversight to ensure the responsible use of genetic data while striking a balance between public safety and the protection of individuals' privacy rights. These findings add to the growing body of evidence regarding the potential benefits of forensic DNA kinship matching as a tool in criminal investigations, suggesting its potential future utilization and legalization.

An inter-laboratory comparison of probabilistic genotyping parameters and evaluation of performance on DNA mixtures from different laboratories.

Probabilistic genotyping (PG) is becoming the preferred standard for evidence interpretation, amongst forensic DNA laboratories, especially those in the United States. Various groups have expressed concern about reliability of PG systems, especially for mixtures beyond two contributors. Studies involving interlaboratory testing of known mixtures have been identified as ways to evaluate the reliability of PG systems. Reliability means different things in different contexts. However, it suffices here to think about it as a mixture of precision and accuracy. We might also consider whether a system is prone to producing misleading results - for example large likelihood ratios (LRs) when the POI is truly not a contributor, or small LRs when the POI is a truly a contributor. In this paper we show that the PG system STRmix™ is relatively unaffected by differences in parameter settings. That is, a DNA mixture that is analyzed in different laboratories using STRmix™ will result in different LRs, but less than 0.05% of these LRs would result in a different, or misleading conclusion as long as the LR is greater than 50. For the purposes of this study, we define LRs assigned using different parameters for the same mixtures as similar if the LR of the true POI is greater than the LRs generated for 99.9% of the general population. These findings are based on an interlaboratory study involving eight laboratories that provided twenty known DNA mixtures of two to four contributors and their individual laboratory STRmix™ parameters. The eight sets of laboratory parameters included differences in STR kits and PCR cycles as well as the peak, stutter, and locus specific amplification efficiency variances.

Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method.

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.

Validation process of automatic DNA extraction from bone material using a new advanced protocol for the EZ2 Connect instrument.

Identification of human remains using genetic methods is an important task of forensic science. DNA markers are proving essential in the identification of unknown human remains. However, environmental factors can lead to poor preservation of DNA, including in bone material. The aim of this study was therefore to compare two methods of DNA isolation from bone material: the traditional organic method and the new protocol using the EZ2 Connect instrument. The study involved three types of bone material, namely molars/premolars, petrous parts of the temporal bone and femurs, all with an estimated PMI of 70-80 years. Importantly, the biological material was obtained from three different environments, categorized as preserving, neutral and degrading, based on basic physico-chemical tests and the potential impact on the bone. The results obtained show that the DNA was best preserved in the petrous bone, followed by the teeth, and the femur. DNA extraction using the EZ2 Connect instrument with a new protocol gave slightly better results for the petrous bone, comparable results for the teeth and worse results for the femur compared to the organic method. Several protocol modifications were tested and optimal conditions for DNA isolation were proposed for the EZ2 protocol. Furthermore, the use of an automated method facilitated the effective accumulation of isolates and increased the chances of successful identification of unknown human remains.

Evaluation of Storage Conditions and the Effect on DNA from Forensic Evidence Objects Retrieved from Lake Water.

DNA analysis of traces from commonly found objects like knives, smartphones, tapes and garbage bags related to crime in aquatic environments is challenging for forensic DNA laboratories. The amount of recovered DNA may be affected by the water environment, time in the water, method for recovery, transport and storage routines of the objects before the objects arrive in the laboratory. The present study evaluated the effect of four storage conditions on the DNA retrieved from bloodstains, touch DNA, fingerprints and hairs, initially deposited on knives, smartphones, packing tapes, duct tapes and garbage bags, and submerged in lake water for three time periods. After retrieval, the objects were stored either through air-drying at room temperature, freezing at -30 °C, in nitrogen gas or in lake water. The results showed that the submersion time strongly influenced the amount and degradation of DNA, especially after the longest submersion time (21 days). A significant variation was observed in success for STR profiling, while mtDNA profiling was less affected by the submersion time interval and storage conditions. This study illustrates that retrieval from water as soon as possible and immediate storage through air-drying or freezing before DNA analysis is beneficial for the outcome of DNA profiling in crime scene investigations.

Targeted recovery of male cells in a male and female same-cell mixture.

DNA mixture deconvolution in the forensic DNA community has been addressed in a variety of ways. "Front-end" methods that separate the cellular components of mixtures can provide a significant benefit over computational methods as there is no need to rely on models with inherent uncertainty to generate conclusions. Historically, cell separation methods have been investigated but have been largely ineffective due to high cost, unreliability, and the lack of proper instrumentation. However, the last decade has given rise to more innovative technology that can target and recover cells more effectively. This study focuses on the development and optimization of a method to selectively label and recover male cells in a mixture of male and female epithelial cells using a Y-chromosome labeling kit with DEPArray™ technology, whereby male cells are labeled and recovered into a single extraction-ready tube. Labeling efficiency was tested using freshly collected and aged buccal swabs where 70%-75% and 38% of male cells were labeled, respectively, with less than 1% false positives. DEPArray™ detection was assessed using single buccal epithelial cells where approximately 80% of labeled cells were identified as male. Mixtures (1:1, 1:10, male to female) yielded profiles that were predominantly single source male or those in which the male component was more easily interpreted. The male-specific labeling method was demonstrated to be both robust and reliable when used on freshly collected cells. While the DEPArray™ meditated detection and recovery had notable limitations, it still improved the interpretation of the male component in same-cell mixtures in more recently collected samples.

Precision touch DNA sampling on plastic bag knots for improved profiling of packer and holder contributions.

In forensic DNA analysis, evidence sampling stands as a pivotal step setting the ground for the quality of the forensic profiling. The collection of touch DNA from objects, when guidelines are scarce or absent, is usually governed by ad hoc decisions based on the available case circumstances. In our laboratory, in the context of illicit drug-related crimes, similar objects are frequently encountered, offering an opportunity for the standardization of evidence treatment. This study aims to develop an effective method for sampling touch DNA from knots on plastic bags. We examine both the exposed and hidden areas of knots, considering the latter as "protected" zones less likely to accumulate biological material during subsequent handling. The study contrasts a single sample method (whole knot surface sampling, Method 1) with dual-sample methods that separate exterior (exposed) and interior (hidden) surfaces of the knot. Notably, our study consistently reveals higher DNA yields from exterior surfaces of the knots as opposed to interior samples. Importantly, our findings demonstrate that utilizing a single sample may produce DNA profiles that are not interpretable, while employing a dual-sample approach may allow for the differentiation between the genetic contributions of the person who tied the knot, the packer, from the person who held the package, the holder. We have refined the dual-sample method to reduce holder DNA in the interior sample while maintaining it on the exterior, also allowing the packer's DNA to be detected on both surfaces. We explore four dual-sample collection methods. Method 2 involves taking the first sample from the exterior and the second from the interior of an untied knot. Method 3 visually differentiates between the original exposed and hidden surfaces for precise sampling. Method 4 employs tools to open the knot for interior sampling. Method 5 uses Diamond dye to highlight cell-free DNA on both surfaces before sampling. In conclusion, this study not only clarifies the complex dynamics of touch DNA transfer and collection on plastic bag knots, but also offers insights into standardizing evidence collection in similar cases.

Developmental validation of the STRSeqTyper122 kit for massively parallel sequencing of forensic STRs.

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.

Targeted enrichment of whole-genome SNPs from highly burned skeletal remains.

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.

Law enforcement use of genetic genealogy databases in criminal investigations: Nomenclature, definition and scope.

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.

Genotype data for 60 SNP genetic markers associated with eye, hair, skin color, ABO blood group, sex, core Y-chromosome haplogroups in Kazakh population.

OBJECTIVES: The collection of genotype data was conducted as an essential part of a pivotal research project with the goal of examining the genetic variability of skin, hair, and iris color among the Kazakh population. The data has practical application in the field of forensic DNA phenotyping (FDA). Due to the limited size of forensic databases from Central Asia (Kazakhstan), it is practically impossible to obtain an individual identification result based on forensic profiling of short tandem repeats (STRs). However, the pervasive use of the FDA necessitates validation of the currently employed set of genetic markers in a variety of global populations. No such data existed for the Kazakhs. The Phenotype Expert kit (DNA Research Center, LLC, Russia) was used for the first time in this study to collect data. DATA DESCRIPTION: The present study provides genotype data for a total of 60 SNP genetic markers, which were analyzed in a sample of 515 ethnic Kazakhs. The dataset comprises a total of 41 single nucleotide polymorphisms (SNPs) obtained from the HIrisPlex-S panel. Additionally, there are 4 SNPs specifically related to the AB0 gene, 1 marker associated with the AMELX/Y genes, and 14 SNPs corresponding to the primary haplogroups of the Y chromosome. The aforementioned data could prove valuable to researchers with an interest in investigating genetic variability and making predictions about phenotype based on eye color, hair color, skin color, AB0 blood group, gender, and biogeographic origin within the male lineage.

Co-amplification of microbial DNA: A novel observation of a misconstrued second peak at locus D7S820.

DNA fingerprinting, a gold standard, is one of the most powerful tool in applied sciences especially helpful in criminal investigation. Entering in advanced era of forensic DNA, profile reading is much trickier than ever. An unusual DNA profile was observed from a nail swab of female brutally murdered in a domestic violence case. At first, DNA profile was misconstrued as heterozygote at locus D7S820 but later, it was confirmed as homozygous from other evidence items submitted in the same case. Subsequent reprocessing of the same sample, from the extraction stage through to DNA profiling and DNA profile form victim's blood, conclusively established that the unusual peak is from a non-specific microbial presence at that locus.