Forensic height estimation using polygenic score in Korean population.

Height is known to be a classically heritable trait controlled by complex polygenic factors. Numerous height-associated genetic variants across the genome have been identified so far. It is also a representative of externally visible characteristics (EVC) for predicting appearance in forensic science. When biological evidence at a crime scene is deficient in identifying an individual, the examination of forensic DNA phenotyping using some genetic variants could be considered. In this study, we aimed to predict 'height', a representative forensic phenotype, by using a small number of genetic variants when short tandem repeat (STR) analysis is hard with insufficient biological samples. Our results not only replicated previous genetic signals but also indicated an upward trend in polygenic score (PGS) with increasing height in the validation and replication stages for both genders. These results demonstrate that the established SNP sets in this study could be used for height estimation in the Korean population. Specifically, since the PGS model constructed in this study targets only a small number of SNPs, it contributes to enabling forensic DNA phenotyping even at crime scenes with a minimal amount of biological evidence. To the best of our knowledge, this was the first study to evaluate a PGS model for height estimation in the Korean population using GWAS signals. Our study offers insight into the polygenic effect of height in East Asians, incorporating genetic variants from non-Asian populations.

Forensic stability evaluation of selected miRNA and circRNA markers in human bloodstained samples exposed to different environmental conditions.

Recently, RNA markers have been used to identify tissue origins of different kinds of body fluids. Herein, circRNA and miRNA markers were carried out to examine the presence or absence of peripheral blood (PB) in bloodstained samples exposed to different external environmental conditions, which mimicked PB samples left at the crime scenes. PB samples were placed on sterile swabs and then exposed to different high temperatures (37°C, 55°C and 95°C) and ultraviolet light irradiation for 0 d, 0.5 d, 1 d, 3 d, and 7 d, ultra-low and low temperatures (-80°C, -20°C, and 4°C) for 30 d, 180 d and 365 d and different kinds of disinfectants. Total RNA was extracted from bloodstained samples under the above different conditions, and the expressions of target RNAs (including miR16-5p, miR451a, circ0000095, and two reference genes RNU6b and 18 S rRNA) were detected by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. Results showed that these selected RNA markers could be successfully measured at all observation points with their unique degradation rates, which exhibited relative stability in degraded bloodstained samples exposed to different environmental conditions. This study provides insights into the applications of these studied miRNA and circRNA markers in forensic science.

Recommendations of the DNA Commission of the International Society for Forensic Genetics (ISFG) on short tandem repeat sequence nomenclature.

The DNA Commission of the International Society for Forensic Genetics (ISFG) has developed a set of nomenclature recommendations for short tandem repeat (STR) sequences. These recommendations follow the 2016 considerations of the DNA Commission of the ISFG, incorporating the knowledge gained through research and population studies in the intervening years. While maintaining a focus on backward compatibility with the CE data that currently populate national DNA databases, this report also looks to the future with the establishment of recommended minimum sequence reporting ranges to facilitate interlaboratory comparisons, automated solutions for sequence-based allele designations, a suite of resources to support bioinformatic development, guidance for characterizing new STR loci, and considerations for incorporating STR sequences and other new markers into investigative databases.

Developmental validation of the AGCU YNFS Y Kit: A new 6-dye multiplex system with 44 Y-STRs and 5 Y-InDels for forensic application.

With the widespread use of the Y chromosome in genetics, a lot of commercially available Y chromosome kits were developed, validated, and applied to forensic science practice. The AGCU YNFS Y Kit is a new Y chromosome system containing forty-four preferred Y short tandem repeats (Y-STRs) and five common Y-InDels. In this study, the AGCU YNFS Y system was validated to verify its performance by following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM). A series of validation experiments included the following parameters: PCR-based studies, sensitivity studies, species specificity studies, stability studies, mixture studies, precision studies, stutter calculation, mutation and statistical analysis, population study, and case samples and degradation studies. The results suggested that appropriately changing PCR amplification conditions did not affect genotyping; the kit had good sensitivity for trace amounts of DNA (0.0625 ng), mixtures of multiple male individuals (minor: major = 1: 9), and three PCR inhibitors (more than 250 µM hematin, 250 ng/µL humic acid and 50 ng/µL tannic acid). The maximum standard deviation of allele size did not exceed 0.1552 reflecting the high accuracy of the system. By this, 87 DNA-confirmed pairs of father-son pairs were also analyzed for mutations. A total of 18 loci were mutated, with mutation rates ranging from 11.5×10-3 to 34.5×10-3 (95% CI 7.2×10-3-97.5×10-3, DYS627 and DYF404S1). In the population study, the haplotype diversity of 87 unrelated individuals was 0.9997, and discrimination capacity was 0.9885. Degradation studies have demonstrated that UV-C light exposure for up to 120 hours has no effect on male blood and semen-vaginal secretion mixtures. However, complete typing could no longer be obtained after 48 hours of UV exposure in single male saliva and in male saliva and female blood mixed samples. Collectively, the AGCU YNFS Y Kit is sensitive and accurate and can play its application value in forensic science practice.

DNA profiling in India: Addressing issues of sample preservation, databasing, marker selection, & statistical approaches.

DNA technology is the gold standard with respect to the identification of individuals from biological evidence. The technology offers the convenience of a universally similar approach and methodology for analysis across the globe. However, the technology has not realised its full potential in India due to the lack of a DNA database and lacunae in sample collection and preservation from the scene of crime and victims (especially those of sexual assault). Further, statistical interpretation of DNA results is non-existent in the majority of cases. Though the latest technologies and developments in the field of DNA analysis are being adopted and implemented,very little has been enacted practically to improve optimise sample collection and preservation. This article discusses current casework scenarios that highlight the pitfalls and ambiguous areas in the field of DNA analysis, especially with respect DNA databases, sampling, andstatistical approaches to genetic data analysis. Possible solutions and mitigation measures are suggested.

Kafka's beautiful eyes: Forensic intelligence utilisation of phenotypic information.

Franz Kafka had beautiful eyes. So striking, that many of the famous author's friends and peers commented on them - but quite variously ('dark', 'brown', 'grey' & 'blue'). Eye colour as perceived by an observer is subjective, being influenced by physiological, environmental, and even sociocultural factors. In a policing context, this does not mean that trait information such as eye colour is not valuable (far from it), but that it must be managed carefully. The Australian Federal Police has recently implemented a forensic DNA phenotyping (FDP, aka. physical trait prediction or PTP) capability, utilising massively parallel sequencing DNA technology to predict an individual's eye colour, biogeographical ancestry and sex from a crime scene sample. This information alone is not itself 'intelligence', but can be used to generate intelligence through holistic analyses undertaken within a transdisciplinary, all-source forensic intelligence (FORINT) framework. FORINT outputs posit abductive propositions typically at the activity/offence level, to provide insight and influence decision making. However, the use of predicted traits requires that they are compared to something; all Australian police databases include fields for physical traits, but no uniform standard is applied across all agencies. Moreover, collection is inconsistent and no automated systems are in place to capture such data systematically. Consider the 'Kafka problem': his peers gave multiply divergent descriptions of his eyes. If a Biology unit had predicted the eye colour of an 'unidentified author' using DNA - how would Kafka be confidently nominated as the contributor? We posit three maxims for law enforcement: (1) To expand the operational utility of forensic science in line with police demands, forensic science should operationalise FDP (e.g. operationally to rank a list of persons of interest, focus lines of enquiry in serious & organised crime, or assist with human remains identification). (2) Such advanced biological techniques are best delivered through an all-source FORINT framework, to maximise opportunities and minimise risk. (3) One cannot pursue techno-scientific advancements in isolation; it is also necessary to influence the operational posture for their implementation. In this paper we explore these issues and provide recommendations relating to (a) police practices, (b) image capture systems, and (c) research opportunities. Phenotypic trait prediction has great potential and can be operationalised effectively through a rigorous FORINT framework. However, there is (continual) work to be done to enhance the operational capabilities that are complementary to - but necessary for - effective forensic science contribution to investigations.

Hungarian legislation regarding implementing a forensic DNA elimination database.

The inception of forensic DNA elimination database represents a pivotal advancement in forensic science, aiming to streamline the process of distinguishing between DNA found at crime scenes and that of individuals involved in the investigation process, such as law enforcement personnel and forensic lab staff. In subsequent phases, once familiarity with the database is achieved by its administrators and other stakeholders, and they have accrued sufficient experience, the possibility of expanding the database to encompass first responders-including firefighters, paramedics, emergency medical technicians, and other emergency services personnel-can be contemplated. Key challenges in managing these databases encompass the grounds for collecting samples, ensuring the integrity of both samples and profiles, along with the duration of retention, access to the database, and the protocols to follow when a match is found in the database. This paper outlines the conceptual and detailed legislative framework in Hungary, where the forensic DNA elimination database was introduced in 2022.

Saliva-derived secondary DNA transfer on fabric: The impact of varying conditions.

This study explored secondary DNA transfer involving saliva, a body fluid often encountered in forensic investigations. Various factors were examined to investigate their potential impact on the transfer of DNA from saliva stains deposited onto common types of fabric (cotton, nylon, and towel). We examined varying types of saliva moisture (wet, dry, and rehydrated) and different types of contact (controlled pressure and active/friction pressure) to quantitatively evaluate how such variables could impact transfer and possible conclusions surrounding saliva-derived DNA deposits. The transfer of DNA was generally least pronounced with more absorbent primary fabrics (cotton and towel materials) while a less absorbent primary fabric (nylon) exhibited a greater propensity for DNA transfer. There were significantly higher amounts of transferred DNA (p < 0.05) observed in wet saliva samples compared to dry and rehydrated saliva samples. Further, the use of active pressure (friction) appeared to result in more DNA transfer overall as compared to controlled pressure contact. Experiments conducted with wet saliva and active pressure (friction) demonstrated the highest likelihood of transfer, with the primary nylon and secondary towel fabric combination demonstrating the greatest average transfer percentage of 94.74 %. The variables explored in this study presented multiple combinations wherein a sufficient amount of DNA (≥ 240 pg total) was transferred to the secondary fabric, making it potentially suitable for STR-PCR amplification in our laboratory. The findings from this study indicate that the type of primary fabric receiving the saliva deposit, the type of saliva moisture, the type of secondary fabric and its moisture type, and the type of contact all have the potential to affect the quantity of DNA transferred and recovered. This study provides empirical data on the ease, and to what extent, DNA from saliva transfers between fabrics and aids DNA activity level evaluations. The significance of this research lies in its contribution to expanding our current understanding of DNA transfer involving saliva within forensic science and criminal investigations.

The advent of forensic DNA databases: It's time to agree on some international governance principles!

National forensic DNA databases are a valuable investigative tool, that have the potential to increase the efficacy of criminal investigations. Their unfettered expansion in recent years raises unsettling ethical issues that require close attention. DNA database expansion threatens the rights to privacy, non-discrimination, and equality, and can undermine public trust in government. This perspective piece relies on data from an international mapping study of Forensic DNA Databases to document the expansion of these databases, highlight the ethical issues they raise, and propose key recommendations for more responsible use of this infrastructure.

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.

Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

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

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

Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing.

In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures. Traditional STR profiles face difficulties in such scenarios. Over recent years, RNA has emerged as a promising biomarker for body fluid identification, and mRNA polymorphism has shown excellent performance in identifying body fluid donors in previous studies. In this study, a massively parallel sequencing assay was developed, encompassing 202 coding region SNPs (cSNPs) from 45 body fluid/tissue-specific genes to identify both body fluid/tissue origin and the respective donors, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin. The specificity was evaluated by examining the single-source body fluids/tissue and revealed that the same body fluid exhibited similar expression profiles and the tissue origin could be identified. For laboratory-generated mixtures containing 2-6 different components and mock case mixtures, the donor of each component could be successfully identified, except for the skin donor. The discriminatory power for all body fluids ranged from 0.997176329 (menstrual blood) to 0.99999999827 (blood). The concordance of DNA typing and mRNA typing for the cSNPs in this system was also validated. This cSNP typing system exhibits excellent performance in mixture deconvolution.

Construction of 13-locus X-STR multiplex PCR system that reinforces Argus X-12 kit discrimination capacity: Application to the Malay population.

Closely linked groups of markers on the X chromosome are very useful for testing complex kinship relationships involving X-STR transmission. The Argus X-12 kit, a unique commercially available kit, can obtain haplotypes of 4 linkage groups (LGs) consisting of 3 markers. Although many population data have been reported for forensic purposes, differences in discrimination ability exist between LG1 and LG2, 3, and 4 in East Asian populations, and the data of this kit would become more useful if the discrimination ability of the latter groups were increased. Therefore, for matches found using this kit for some linkage group data, then to increase the identification ability, we additionally introduced 13 X-STR loci and established a method allowing comparison using data from 25 loci. The 13X-STRs add two locus data to each of LG2, 3, and 4, and also add two closely linked group (CLG) data between LG2 and 3 and LG3 and 4 in one multiplex PCR. Assessment of this method for a Malay population for which data by Argus X-12 had already been reported showed that the frequencies of distinct haplotypes in LG2, 3, and 4 were increased by 33.0-42.6 %, and frequencies of unique haplotypes increased by 45.4-59.2 %. The respective haplotype diversity values of the additional 3-locus and 4-locus CLGs were 0.9838 and 0.9939, which helps to improve discriminatory power and to predict recombination locations on the X chromosome. Although we have been testing these loci with Japanese subjects, this system would also be useful for the Malay population.

The Usefulness of qPCR Data for Sample Pre-Assessment and Interpretation of Genetic Typing Results.

DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction's nature, qPCR assays may be subjected to the same stochastic effects of traditional PCR for low-input concentrations. The study aims to evaluate the precision of the PowerQuant® (Promega) kit assay measurements and the degree of variability for DNA templates falling below the optimal threshold of the PowerPlex® ESX-17 Fast STR typing kit (Promega). Five three-fold dilutions of the 2800 M control DNA (Promega) were set up. Each dilution (concentrations: 0.05, 0.0167, 0.0055, 0.00185, and 0.000617 ng/µL) was quantified and amplified in four replicates. Variability for qPCR results, STR profile completeness, and EPGs' peak height were evaluated. The qPCR-estimated concentration of casework samples was correlated with profile completeness and peak intensity, to assess the predictive value of qPCR results for the successful STR typing of scarce samples. qPCR was subjected to stochastic effects, of which the degree was inversely proportional to the initial input template. Quantitation results and the STR profile's characteristics were strongly correlated. Due to the intrinsic nature of real casework samples, a qPCR-derived DNA concentration threshold for correctly identifying probative STR profiles may be difficult to establish. Quantitation data may be useful in interpreting and corroborating STR typing results and for clearly illustrating them to the stakeholders.

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.

An Assessment of the Performance Limitations of the Integrated QuantifilerTM Trio-HRM Assay: A Forensic Tool Designed to Identify Mixtures at the Quantification Stage.

Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, we integrated a high-resolution melt (HRM) mixture screening assay into the qPCR step of the forensic workflow, producing the integrated QuantifilerTM Trio-HRM assay. The assay, when coupled with a prediction tool, allowed for 75.0% accurate identification of the contributor status of a sample (single source vs. mixture). To elucidate the limitations of the developed qPCR-HRM assay, developmental validation studies were conducted assessing the reproducibility and samples with varying DNA ratios, contributors, and quality. From this work, it was determined that the integrated QuantifilerTM Trio-HRM assay is capable of accurately identifying mixtures with up to five contributors and mixtures at ratios up to 1:100. Further, the optimal performance concentration range was found to be between 0.025 and 0.5 ng/µL. With these results, evidentiary-like DNA samples were then analyzed, resulting in 100.0% of the mixture samples being accurately identified; furthermore, every time a sample was predicted as a single source, it was true, giving confidence to any single-source calls. Overall, the integrated QuantifilerTM Trio-HRM assay has exhibited an enhanced ability to discern mixture samples from single-source samples at the qPCR stage under commonly observed conditions regardless of the contributor's sex.

Improved individual identification in DNA mixtures of unrelated or related contributors through massively parallel sequencing.

DNA mixtures are a common sample type in forensic genetics, and we typically assume that contributors to the mixture are unrelated when calculating the likelihood ratio (LR). However, scenarios involving mixtures with related contributors, such as in family murder or incest cases, can also be encountered. Compared to the mixtures with unrelated contributors, the kinship within the mixture would bring additional challenges for the inference of the number of contributors (NOC) and the construction of probabilistic genotyping models. To evaluate the influence of potential kinship on the individual identification of the person of interest (POI), we conducted simulations of two-person (2 P) and three-person (3 P) DNA mixtures containing unrelated or related contributors (parent-child, full-sibling, and uncle-nephew) at different mixing ratios (for 2 P: 1:1, 4:1, 9:1, and 19:1; for 3 P: 1:1:1, 2:1:1, 5:4:1, and 10:5:1), and performed massively parallel sequencing (MPS) using MGIEasy Signature Identification Library Prep Kit on MGI platform. In addition, in silico simulations of mixtures with unrelated and related contributors were also performed. In this study, we evaluated 1): the MPS performance; 2) the influence of multiple genetic markers on determining the presence of related contributors and inferring the NOC within the mixture; 3) the probability distribution of MAC (maximum allele count) and TAC (total allele count) based on in silico mixture profiles; 4) trends in LR values with and without considering kinship in mixtures with related and unrelated contributors; 5) trends in LR values with length- and sequence-based STR genotypes. Results indicated that multiple numbers and types of genetic markers positively influenced kinship and NOC inference in a mixture. The LR values of POI were strongly dependent on the mixing ratio. Non- and correct-kinship hypotheses essentially did not affect the individual identification of the major POI; the correct kinship hypothesis yielded more conservative LR values; the incorrect kinship hypothesis did not necessarily lead to the failure of POI individual identification. However, it is noteworthy that these considerations could lead to uncertain outcomes in the identification of minor contributors. Compared to length-based STR genotyping, using sequence-based STR genotype increases the individual identification power of the POI, concurrently improving the accuracy of mixing ratio inference using EuroForMix. In conclusion, the MGIEasy Signature Identification Library Prep kit demonstrated robust individual identification power, which is a viable MPS panel for forensic DNA mixture interpretations, whether involving unrelated or related contributors.

Identification of individuals from low template blood samples using whole transcriptome shotgun sequencing.

Biological trace samples consisting of very few cells pose a challenge to conventional forensic genetic DNA analysis. RNA may be an alternative to DNA when handling low template samples. Whereas each cell only contains two copies of an autosomal DNA segment, the transcriptome retains much of the genomic variation replicated in abundant RNA fragments. In this study, we describe the development of a prototype RNA-based SNP selection set for forensic human identification from low template samples (50 pg gDNA). Whole blood from a subset of the Danish population (41 individuals) and blood stains subjected to degradation at room temperature for up to two weeks were analysed by whole transcriptome shotgun sequencing. Concordance was determined by DNA genotyping with the Infinium Omni5-4 SNP chip. In the 100 protein-coding genes with the most reads, 5214 bi-allelic SNPs with gnomAD minor allele frequencies > 0.1 in the African/African American, East Asian, and (non-Finnish) European populations were identified. Of these, 24 SNPs in 21 genes passed screening in whole blood and degraded blood stains, with a resulting mean match probability of 4.5 ∙ 10-9. Additionally, ancestry informative SNPs and SNPs in genes useful for body fluid identification were identified in the transcriptome. Consequently, shotgun sequencing of RNA from low template samples may be used for a vast host of forensic genetics purposes, including simultaneous human and body fluid identification, leading to direct donor identification in the identified body fluid.