Joint application of A-InDels and miniSTRs for forensic personal, full and half sibling identifications, and genetic differentiation analyses in two populations from China.

BACKGROUND: Previously, a novel multiplex system of 64 loci was constructed based on capillary electrophoresis platform, including 59 autosomal insertion/deletions (A-InDels), two Y-chromosome InDels, two mini short tandem repeats (miniSTRs), and an Amelogenin gene. The aim of this study is to evaluate the efficiencies of this multiplex system for individual identification, paternity testing and biogeographic ancestry inference in Chinese Hezhou Han (CHH) and Hubei Tujia (CTH) groups, providing valuable insights for forensic anthropology and population genetics research. RESULTS: The cumulative values of power of discrimination (CDP) and probability of exclusion (CPE) for the 59 A-InDels and two miniSTRs were 0.99999999999999999999999999754, 0.99999905; and 0.99999999999999999999999999998, 0.99999898 in CTH and CHH groups, respectively. When the likelihood ratio thresholds were set to 1 or 10, more than 95% of the full sibling pairs could be identified from unrelated individual pairs, and the false positive rates were less than 1.2% in both CTH and CHH groups. Biogeographic ancestry inference models based on 35 populations were constructed with three algorithms: random forest, adaptive boosting and extreme gradient boosting, and then 10-fold cross-validation analyses were applied to test these three models with the average accuracies of 86.59%, 84.22% and 87.80%, respectively. In addition, we also investigated the genetic relationships between the two studied groups with 33 reference populations using population statistical methods of FST, DA, phylogenetic tree, PCA, STRUCTURE and TreeMix analyses. The present results showed that compared to other continental populations, the CTH and CHH groups had closer genetic affinities to East Asian populations. CONCLUSIONS: This novel multiplex system has high CDP and CPE in CTH and CHH groups, which can be used as a powerful tool for individual identification and paternity testing. According to various genetic analysis methods, the genetic structures of CTH and CHH groups are relatively similar to the reference East Asian populations.

Establishment and Application of a 42-plex Microhaplotype Assay in Forensic Genetics.

OBJECTIVES: To establish and forensically verify a 42 microhaplotypes (mircohaps, MHs) multiplex assay system based on next-generation sequencing (NGS), and to explore the application value of this system in the practice of forensic genetics. METHODS: A total of 42 highly polymorphic MHs were selected from previous studies, and sequenced by the MiSeq FGxTM platform to verify the repeata-bility, sensitivity, specificity, stability, and mixture analysis ability of the detection system. Through population genetic investigation of 102 unrelated Chinese Han individuals in Liyang City, Jiangsu Province, China, the application value of this system in forensic genetics was evaluated. RESULTS: The sequencing repeatability of the 42-plex MHs assay was 100% and the sensitivity was as low as 0.062 5 ng. The system had the ability to withstand the interference of indigo (≤2 500 ng/µL), humic acid (≤9 ng/µL), hemoglobin(≤20 µmol), and urea (≤200 ng/µL) and to detect mixtures of 2 people (1∶19), 3 people (1∶1∶9) and 4 people (1∶1∶1∶9). Based on 102 individual data, the combined power of discrimination and the combined power of exclusion were 1-3.45×10-30 and 1-3.77×10-11, respectively, and the average effect value of alleles was 2.899. CONCLUSIONS: The 42-plex MHs assay was successfully established in this study and this system has high repeatability and sensitivity, good anti-jamming ability and mixture analysis ability. The 42 MHs are highly polymorphism and have good application value in individual identification and paternity testing.

Mass spectrometry-based proteomic analysis of biological stains identifies body fluids specific markers.

The identification of biological stains and their tissue resource is an important part of forensic research. Current methods suffer from several limitations including poor sensitivity and specificity, trace samples, and sample destruction. In this study, we profiled the proteomes of menstrual blood, peripheral blood, saliva, semen, and vaginal fluid with mass spectrometry technology. Tissue-enhanced and tissue-specific proteins of each group have been proposed as potential biomarkers. These candidate proteins were further annotated and screened through the combination with the Human Protein Atlas database. Our data not only validates the protein biomarkers reported in previous studies but also identifies novel candidate biomarkers for human body fluids. These candidates lay the foundation for the development of rapid and specific forensic examination methods.

A novel 193-plex MPS panel integrating STRs and SNPs highlights the application value of forensic genetics in individual identification and paternity testing.

Massively parallel sequencing (MPS) has emerged as a promising technology for targeting multiple genetic loci simultaneously in forensic genetics. Here, a novel 193-plex panel was designed to target 28 A-STRs, 41 Y-STRs, 21 X-STRs, 3 sex-identified loci, and 100 A-SNPs by employing a single-end 400 bp sequencing strategy on the MGISEQ-2000™ platform. In the present study, a series of validations and sequencing of 1642 population samples were performed to evaluate the overall performance of the MPS-based panel and its practicality in forensic application according to the SWGDAM guidelines. In general, the 193-plex markers in our panel showed good performance in terms of species specificity, stability, and repeatability. Compared to commercial kits, this panel achieved 100% concordance for standard gDNA and 99.87% concordance for 14,560 population genotypes. Moreover, this panel detected 100% of the loci from 0.5 ng of DNA template and all unique alleles at a 1:4 DNA mixture ratio (0.2 ng minor contributor), and the applicability of the proposed approach for tracing and degrading DNA was further supported by case samples. In addition, several forensic parameters of STRs and SNPs were calculated in a population study. High CPE and CPD values greater than 0.9999999 were clearly demonstrated and these results could be useful references for the application of this panel in individual identification and paternity testing. Overall, this 193-plex MPS panel has been shown to be a reliable, repeatable, robust, inexpensive, and powerful tool sufficient for forensic practice.

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

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

Investigative use of human environmental DNA in forensic genetics.

Individuals leave behind traces of their DNA wherever they go. DNA can be transferred to surfaces and items upon touch, can be released into the air, and may be deposited in indoor dust. The mere presence of individuals in a location is sufficient to facilitate either direct or indirect DNA transfer into the surrounding environment. In this study, we analyzed samples recovered from commonly touched surfaces such as light switches and door handles in an office environment. We evaluated two different methods to isolate DNA and co-extract DNA and RNA from the samples. DNA profiles were compared to the references of the inhabitants of the different locations and were analyzed taking into consideration the type of sampled surface, sampling location and information about the activities in a room during the sampling day. Results from DNA samples collected from surfaces were also compared to those from air and dust samples collected in parallel from the same areas. We characterized the amount and composition of DNA found on various surfaces and showed that surface DNA sampling can be used to detect occupants of a location. The results also indicate that combining information from environmental samples collected from different DNA sources can improve our understanding of DNA transfer events in an indoor setting. This study further demonstrates the potential of human environmental DNA as an investigative tool in forensic genetics.

Epigenetic-based age prediction in blood samples: Model development.

Aging is a complex process influenced by genetic, epigenetic, and environmental factors that lead to tissue deterioration and frailty. Epigenetic mechanisms, such as DNA methylation, play a significant role in gene expression regulation and aging. This study presents a new age estimation model developed for the Turkish population using blood samples. Eight CpG sites in loci TOM1L1, ELOVL2, ASPA, FHL2, C1orf132, CCDC102B, cg07082267, and RASSF5 were selected based on their correlation with age. Methylation patterns of these sites were analyzed in blood samples from 100 volunteers, grouped into age categories (20-35, 36-55, and ≥56). Sensitivity analysis indicated a reliable performance with DNA inputs ≥1 ng. Statistical modeling, utilizing Multiple Linear Regression, underscores the reliability of the primary 6-CpG model, excluding cg07082267 and TOM1L1. This model demonstrates strong correlations with chronological age (r = 0.941) and explains 88% of the age variance with low error rates (MAE = 4.07, RMSE = 5.73 years). Validation procedures, including a training-test split and fivefold cross-validation, consistently confirm the model's accuracy and consistency. The study indicates minimal variation in error scores across age cohorts and no significant gender differences. The developed model showed strong predictive accuracy, with the ability to estimate age within certain prediction intervals. This study contributes to the age prediction by using DNA methylation patterns, which can have disparate applications, including forensic and clinical assessments.

Predicting age from blood by droplet digital PCR using a set of three DNA methylation markers.

Evaluation of DNA methylation (DNAm) patterns is a promising tool for age estimation. The duplex droplet digital PCR (ddPCR) method has been recently investigated for DNAm evaluation, revealing to be a potential methodology for DNAm evaluation and molecular age estimation. In this study, we evaluated DNAm levels of CpGs located at the three age-associated genes ELOVL2, FHL2 and PDE4C using ddPCR to develop an age prediction model. Blood-derived DNA samples from 58 healthy individuals (42 women and 16 men; aged 1-93 years old) were submitted to bisulfite conversion followed by ddPCR using dual-labeled probes targeting methylated and unmethylated DNA sequences. Simple linear regression statistics revealed a strong correlation between DNAm levels and chronological age for FHL2 (R = 0.948; P = 1.472 × 10-29) and PDE4C (R = 0.819; P = 3.917 × 10-15), addressing only one CpG for each gene. For the ELOVL2 gene, evaluating five CpG sites in simultaneous, revealed a strong age correlation (R = 0.887; P = 2.099 × 10-20) in a simple linear regression statistics and very strong age correlation (R = 0.926; P = 2.202 × 10-25) when using quadratic regression statistics. The multivariable regression analysis, using methylation information captured on ELOVL2 (squared), FHL2 and PDE4C genes, revealed a very strong age correlation (R = 0.970; P = 5.356 ×10-33), explaining 93.7 % of age variance, displaying a mean absolute deviation (MAD) between chronological and predicted age of 4.657 years (RMSE = 6.044). We postulate that the ddPCR method should be further investigated for DNAm-based age prediction, because it is a relatively simple and an accurate method that can be routinely used in forensic laboratories for testing a few numbers of markers.

Forensic characteristics and genetic structure of the Chinese Tibetan population revealed by 38 X-chromosomal InDel loci.

With the increasing importance of X-chromosome (Chr-X) genotyping in kinship identification, the exploitation of X chromosome genetic marker multiplex kits is increasing. The Human X-InDels amplification kit is a novel developed system which contained 38 X-chromosomal Insertion/deletion markers (X-InDels) and Amelogenin. Herein, we investigated the genetic diversity of the 38 X-InDels in the Tibetan ethnic minority (n = 792) from seven regions and evaluated the application potential of this novel panel. The rs16368 was the least variable locus, whereas the most polymorphic locus was the rs59605609 in Tibetan population. We confirmed three linkage groups with the haplotype diversities ranged from 0.5032 to 0.5976. The overall combined power of discrimination (PD) in males and females were 0.999999999582066 and 0.999999999999993, respectively. And the overall combined mean exclusion chance (MEC) values were not lower than 0.999125526990159. In addition, we explored the genetic relationships among the Tibetans in seven different regions via series of population comparison analyses, finding that the genetic relationship between the Ngari Tibetan and Chamdo Tibetan was the farthest, which was consistent with geographical distribution.

Forensic parameters and population analysis of 21 autosomal STR loci in the Wuhu Han population from Anhui Province, East China.

BACKGROUND: At present, there are no available genetic data on the AGCU EX22 Kit from the Wuhu Han population. AIM: This study investigates the applicability of the AGCU EX22 kit, designed for the Chinese population for forensic analysis and population genetics of the Wuhu Han population. SUBJECTS AND METHODS: Bloodstains from 1565 unrelated healthy individuals in Wuhu city, Anhui Province, were collected for analysis. The AGCU EX22 kit was used for amplification, and capillary electrophoresis was used to separate the amplification products. Allele frequencies and forensic parameters were determined. The Wuhu Han population was compared to 10 reference populations through genetic distance, a phylogenetic neighbor-joining tree and principal component analysis. RESULTS: In total, 281 alleles and 1187 genotypes were observed. No significant deviations from Hardy-Weinberg equilibrium at any locus were found after Bonferroni's correction. The 21 autosomal short tandem repeat (STR) genetic markers exhibited high informativeness and polymorphism. The cumulative power of discrimination and power of exclusion were 0.999999999999999999999999913380 and 0.999999996752339, respectively. Population comparisons revealed a genetic affinity between Wuhu Han and southern Han populations, except for the Guangdong Han population, which aligned with the traditional geographical division in China. CONCLUSION: The AGCU EX22 Kit, containing 21 STR loci, is suitable for forensic application and population genetics studies in the Wuhu Han population.

Sequence-based population structure, relatedness, and inbreeding estimates for forensic autosomal STR markers.

Population data have become available for sequence data to aid forensic investigations and prepare the forensic community in the move towards implementing NGS methods. This comes with a need for updated population genetic parameters estimates to allow DNA evidence evaluations using sequence data. Initial work has been done on a small sample and here we expand this work by providing estimates of population structure and relatedness for autosomal STR data generated by sequencing technologies. We also discuss the effect of inbreeding on forensic calculations and discuss why the use of genotypic-based estimates may be preferred over allelic-based estimates.

Assessing the usefulness of Raman spectroscopy and lipid analysis of decomposed human bones in forensic genetics and molecular taphonomy.

Bones are among the structures most likely to be recovered after death. However, the low quantity of preserved DNA and complex processing from sample to DNA profile make forensic DNA analysis of bones a challenging task. Raman spectroscopy and gas chromatography-mass spectrometry (GC/MS), have the potential to be useful as screening tools for DNA analysis and in decomposition studies. The objective of this research was to assess the usefulness of such molecular investigations. Femur samples collected from 50 decomposing human bodies were subjected to Raman spectroscopy and GC/MS. Assessment of nuclear DNA quantity and short tandem repeat (STR) genotyping efficiency were also performed. Raman parameters (crystallinity, carbonate-to-phosphate ratio, mineral-to-matrix ratio) and detected lipids were recorded. Background fluorescence proved problematic for Raman analysis of forensic bones. Regardless, it was not associated with less preserved DNA or less detected STR alleles. Fatty acids, hydrocarbons, and five types of fatty acid methyl esters (FAMEs) were detected. The main phosphate peak position in Raman spectra was significantly correlated with preserved DNA (p = 0.03713), while significantly more STR alleles were detected in bones containing methyl hexadecenoate (p = 0.04236). Detection of FAMEs in the bone matrix suggests a reaction between methanol produced by bacteria and free fatty acids, which are not associated with the level of preservation of endogenous DNA. The techniques assessed have shown to be useful in molecular taphonomy studies and forensic genetics.

Expanding the scope of methylation-sensitive restriction enzyme (MSRE) PCR for forensic identification of body fluids through the novel use of methylation-dependent restriction enzymes (MDRE) and the combination of autosomal and Y-chromosomal markers.

Methylation-sensitive/-dependent restriction enzyme (MSRE/MDRE) PCR can be performed to detect hypomethylated or hypermethylated CpG sites. With the combined use of different tissue-specific CpG markers, MSRE/MDRE-PCR leads to tissue-specific methylation patterns (TSMPs), enabling the correlation of DNA samples to their source tissue. MSRE/MDRE assays can use the same platform as forensic STR typing and offer many advantages in the field of forensic body fluid detection. In the present study, we aimed to establish MSRE assays for the detection of blood, saliva, vaginal secretion, and semen, using markers from literature and from our own database search. We designed two different MSRE test-sets, which include two novel Y-chromosomal non-semen markers, and enable differentiation between female and male non-semen samples. Furthermore, we established an MSRE/MDRE semen approach, which includes only Y-chromosomal non-semen and semen markers. This Y-semen multiplex PCR utilizes the novel combination of the methylation-sensitive enzyme SmaI and the methylation-dependent enzyme GlaI, which enables more sensitive detection of male body fluids within male/female DNA mixtures. Our validation tests confirmed that MSRE/MDRE assays exhibit high sensitivity, similar to that of STR typing.

Spitting in the wind?-The challenges of RNA sequencing for biomarker discovery from saliva.

Forensic trace contextualization, i.e., assessing information beyond who deposited a biological stain, has become an issue of great and steadily growing importance in forensic genetic casework and research. The human transcriptome encodes a wide variety of information and thus has received increasing interest for the identification of biomarkers for different aspects of forensic trace contextualization over the past years. Massively parallel sequencing of reverse-transcribed RNA ("RNA sequencing") has emerged as the gold standard technology to characterize the transcriptome in its entirety and identify RNA markers showing significant expression differences not only between different forensically relevant body fluids but also within a single body fluid between forensically relevant conditions of interest. Here, we analyze the quality and composition of four RNA sequencing datasets (whole transcriptome as well as miRNA sequencing) from two different research projects (the RNAgE project and the TrACES project), aiming at identifying contextualizing forensic biomarker from the forensically relevant body fluid saliva. We describe and characterize challenges of RNA sequencing of saliva samples arising from the presence of oral bacteria, the heterogeneity of sample composition, and the confounding factor of degradation. Based on these observations, we formulate recommendations that might help to improve RNA biomarker discovery from the challenging but forensically relevant body fluid saliva.

DNA methylation-based age estimation and quantification of the degradation levels of bisulfite-converted DNA.

DNA methylation modifications are known to influence epigenetic phenomena and have been a focus of forensic science research for some time. Degraded DNA after bisulfite treatment is widely used in DNA methylation analysis. In this study, we analyzed methylation levels at 12 CpG sites of four selected genomic regions by pyrosequencing after bisulfite treatment. DNA was extracted from buccal swab samples collected from 102 Japanese individuals who were 21-77 years old. We also developed a simple method to quantify the degradation levels of bisulfite-converted DNA by real-time PCR, and evaluated the effect of DNA degradation on age estimation. We found that the methylation levels and chronological ages were highly correlated in the four selected regions, and the mean absolute deviation (MAD) between chronological and estimated ages was low at 3.88 years. These results indicated that pyrosequencing analysis at the 12 CpGs was useful for age estimation in the Japanese population. To develop a sensitive quantification method, we analyzed the amplification efficiency of short and long fragments from 10 regions by real-time PCR. The amplification efficiency was highest for CCDC102B, and the degradation levels of bisulfite-converted DNA for the 102 samples were categorized as moderately or heavily degraded. For the younger age groups (20-49 years), the MADs were lower for moderately degraded DNA than they were for heavily degraded DNA. This finding indicates that degradation levels affected the accuracy of age estimation in most of the samples; the exception was the samples from the 50-77 years age group.

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids.

Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)-qPCR duplex targeting miR-891a and let-7g was validated, and miR-891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow.

An mRNA profiling assay incorporating coding region InDels for body fluid identification and the inference of the donor in mixed samples.

Biological traces discovered at crime scenes hold significant significance in forensic investigations. In cases involving mixed body fluid stains, the evidentiary value of DNA profiles depends on the type of body fluid from which the DNA was obtained. Recently, coding region polymorphism analysis has proved to be a promising method for directly linking specific body fluids to their respective DNA contributors in mixtures, which may help to avoid "association fallacy" between separate DNA and RNA evidence. In this study, we present an update on previously reported coding region Single Nucleotide Polymorphisms (cSNPs) by exploring the potential application of coding region Insertion/Deletion polymorphisms (cInDels). Nine promising cInDels, selected from 70 mRNA markers based on stringent screening criteria, were integrated into an existing mRNA profiling assay. Subsequently, the body fluid specificity of our cInDel assay and the genotyping consistency between complementary DNA (cDNA) and genomic DNA (gDNA) were examined. Our study demonstrates that cInDels can function as important multifunctional genetic markers, as they provide not only the ability to confirm the presence of forensically relevant body fluids, but also the ability to associate/dissociate specific body fluids with particular donors.

Review: Wildlife forensic genetics-Biological evidence, DNA markers, analytical approaches, and challenges.

Wildlife-related crimes are the second most prevalent lawbreaking offense globally. This illicit trade encompasses hunting, breeding and trafficking. Besides diminishing many species and their habitats and ecosystems, hindering the economic development of local communities that depend on them, undermining the rule of law and financing terrorism, various cross-species transmissions (zoonoses) of pathogens, including COVID-19, can be attributed to wildlife crimes. Wildlife forensics applies interdisciplinary scientific analyses to support law enforcement in investigating wildlife crimes. Its main objectives are to identify the taxonomic species in question, determine if a crime has been committed, link a suspect to the crime and support the conviction and prosecution of the perpetrator. This article reviews wildlife crime and its implications, wildlife forensic science investigation, common forms of wildlife biological evidence, including DNA, wildlife DNA techniques and challenges in wildlife forensic genetics. The article also reviews the contributions of genetic markers such as short tandem repeat (STR) and mitochondrial DNA (mtDNA) markers, which provide the probative genetic data representing the bulk of DNA evidence for solving wildlife crime. This review provides an overview of wildlife DNA databases, which are critical for searching and matching forensic DNA profiles and sequences and establishing how frequent forensic DNA profiles and sequences are in a particular population or geographic region. As such, this review will contain an in-depth analysis of the current status of wildlife forensic genetics, and it will be of general interest to wildlife and conservation biologists, law enforcement officers, and academics interested in combating crimes against wildlife using animal forensic DNA methods.

A proof-of-principle study: The potential application of MiniHap biomarkers in ancestry inference based on the QNome nanopore sequencing.

Haplotyped SNPs convey forensic-related information, and microhaplotypes (MHs), as the most representative of this kind of marker, have proved the potential value for human forensics. In recent years, nanopore sequencing technology has developed rapidly, with its outstanding ability to sequence long continuous DNA fragments and obtain phase information, making the detection of longer haplotype marker possible. In this proof-of-principle study, we proposed a new type of forensic marker, MiniHap, based on five or more SNPs within a molecular distance less than 800 bp, and investigated the haplotype data of 56 selected MiniHaps in five Chinese populations using the QNome nanopore sequencing. The sequencing performance, allele (haplotype) frequencies, forensic parameters, effective number of alleles (Ae), and informativeness (In) were subsequently calculated. In addition, we performed principal component analysis (PCA), phylogenetic tree, and structure analysis to investigate the population genetic relationships and ancestry components among the five investigated populations and 26 worldwide populations. MiniHap-04 exhibited remarkable forensic efficacy, with 148 haplotypes reported and the Ae was 66.9268. In addition, the power of discrimination (PD) was 0.9934, the probability of exclusion (PE) was 0.9898, and the In value was 0.7893. Of the 56 loci, 85.71% had PD values above 0.85, 66.07% had PE values above 0.54, 67.86% had Ae values over 7.0%, and 55.36% were with In values above 0.2 across all samples, indicating that most of the MiniHaps are suitable for individual identification, paternity testing, mixture deconvolution, and ancestry inference. Moreover, the results of PCA, phylogenetic tree and structure analysis demonstrated that this MiniHap panel had the competency in continental population ancestry inference, but the differentiation within intracontinental/linguistically restricted subpopulations was not ideal. Such findings suggested that the QNome device for MiniHap detection was feasible and this novel marker has the potential in ancestry inference. Yet, the establishment of a more comprehensive database with sufficient reference population data remains necessary to screen more suitable MiniHaps.

Operationalisation of the ForenSeq® Kintelligence Kit for Australian unidentified and missing persons casework.

Single nucleotide polymorphism (SNP) genotyping technologies can generate investigative leads for human remains identification, including estimation of biological sex, biogeographical ancestry (BGA), externally visible characteristics (EVCs), identity, uniparental lineage and extended kinship. The ForenSeq® Kintelligence Kit provides forensic laboratories with the ability to apply this suite of genetic tools to forensic samples using one panel targeting 10,230 SNPs (including 56 ancestry-informative, 24 phenotype-informative, 94 identity-informative, 106 X chromosome, 85 Y chromosome and 9867 kinship-informative SNPs) sequenced on the MiSeq FGx® Sequencing System. The ForenSeq® Kintelligence Kit has been internally validated, optimised and operationalised by the Australian Federal Police National DNA Program for Unidentified and Missing Persons (AFP Program) for coronial casework. The internal validation was conducted according to the Scientific Working Group on DNA Analysis Methods guidelines (excluding mixture analysis), focussing on sample types typically encountered in human remains identification casework, such as bones, teeth, nail, blood and hair. The workflow was optimised for a high throughput library preparation and sequencing workflow, and additional analytical thresholds were developed to improve genotyping accuracy for low DNA input samples. Additionally, the genetic intelligence generated from the kit was compared to the self-declared biological sex, EVCs and BGA of the DNA donors to assess concordance. The kit was able to produce high quality SNP profiles from 1.0 ng down to 0.1 ng of DNA, with high repeatability and reproducibility, and minimal background noise. The prediction accuracy for biological sex (95%), hair colour (58%), eye colour (74%) and BGA inferences (consistent: 74%; partially consistent: 10%; inconclusive: 16%) was determined based on self-declared data. Additionally, SNP profiles from a volunteer family group of ten related individuals were uploaded to GEDmatch PRO™ to assess kinship accuracy. The kit was capable of detecting (97%) and accurately classifying (90%) genetic relationships spanning from first to fifth degree. The Kintelligence Kit provides the AFP Program with a robust and reliable genetic intelligence tool for unidentified and missing persons investigations, which has been designed to sequence multiple challenging samples in a single multiplexed assay using existing laboratory instrumentation.