Feasibility study of a single-primer extension-based microhaplotype NGS system.

DNA degradation has been a thorny problem in forensic science. Shortening the amplicon length of the genetic markers improves the analysis of degraded DNA effectively. Microhaplotype (MH) has been proposed as a potential genetic marker that can be used for degraded DNA analysis. In the present study, a 146-plex MH-next-generation sequencing (NGS) system with an average Ae of 6.876 was constructed. Unlike other MH studies, a single-primer extension (SPE)-based NGS library preparation method was used to improve the detection of MH markers for degraded DNA. SPE employs a locus-specific and universal primer to amplify target fragments, reducing the necessity for complete fragment sequences. SPE might effectively mitigate the impact of degradation on amplification. However, SPE produces amplicons of varying lengths, posing challenges in allele calling for SPE-NGS data. To address this issue, this study proposed a flexible allele-calling strategy to improve amplicon detection. In addition, this study evaluated the forensic efficacy of the system using 12 low-template samples (from 1 ng to 7.8 pg), 10 mock-degraded DNA with various degrees of degradation, and 8 forensic casework samples. When the template is as low as 7.8 pg, our system can accurately detect at least 37 loci and achieves a random match probability (RMP) of 10-30 using the complete allele-calling strategy. Eighty-two loci can be detected, and RMP can reach 10-54 using a flexible allele-calling strategy. After 150 min of 98°C treatment, 36 loci can still be detected, and an RMP of 10-5 can be obtained using the flexible allele-calling strategy. Furthermore, the number of single nucleotide polymorphism detected at different DNA amounts and degradation levels suggests that the SPE method combined with a flexible allele-calling strategy is effective.

Screening a new set of microhaplotypes in exonic regions for sample identity testing and paternity testing during whole exome sequencing analysis.

Whole exome sequencing (WES) is widely used in clinical diagnosis. Before obtaining an accurate diagnosis, it is essential to conduct sample identity testing and paternity testing on trio samples. Currently, there is a lack of optimal genetic markers for these purposes, with limited literature available in this area. Microhaplotypes (MHs) are promising genetic markers due to their high polymorphism, low mutation rate, short amplified fragments, absence of stutter and amplification bias. These characteristics make them suitable for sample tracking and paternity testing during WES analysis. In this study, we screened out a set of polymorphic MHs in exonic regions for the above purposes. The results showed that the power of discrimination (PD) and probability of exclusion (PE) of this set of markers ranged from 0.2682 to 0.8878 and 0.0178 to 0.4583, respectively. Both the cumulative power of discrimination (CPD) and cumulative probability of exclusion (CPE) exceeded 0.999999, indicating the great value of these markers in paternity testing and individual identification in the study population. However, these markers had the effective number of alleles (Ae) values ranging from 1.1784 to 3.8727 (average 2.1805) and informativeness (In) values ranging from 0.0151 to 0.2209 (average 0.0766), showing limited value in DNA mixture analysis and biogeographical ancestry inference.

DIP-microhaplotypes: new markers for detection of unbalanced DNA mixtures.

The identification of a suspect in a degraded and unbalanced DNA mixture has been a challenge for the standard short tandem repeat polymorphisms (STR) typing. Several methods have been introduced to solve this problem, such as DIP-STR, DIP-SNP, and SNP-STR markers. In this study, we proposed DIP-microhaplotype (deletion/insertion linked a chain of SNPs) as a kind of new genetic marker to type the unbalanced and degraded DNA mixture. We established the detection method with ten DIP-microhaplotype markers including 26 SNPs using allele-specific multiplex PCR followed by SNaPshot assay. This novel compound marker allows us to detect the minor DNA with a sensitivity of 1:100 to 1:1000 in a DNA mixture of any gender. Most of the DIP-microhaplotype markers had a relatively high probability of informative alleles with an average informative value (I value) of 0.308. In all, we proposed DIP-microhaplotype as a novel type of DNA marker for the detection of minor contributor from unbalanced DNA mixtures. Due to their inherent shorter length, higher polymorphism, and sensitivity, DIP-microhaplotypes are promising markers for the examination of the degraded and unbalanced mixtures in forensic stains or clinical chimeras.

Identification and sequencing of 59 highly polymorphic microhaplotypes for analysis of DNA mixtures.

Mixture detection remains one of the major challenges within a forensic science context. In recent years, microhaplotypes were proposed to have great potential in mixture detection, although many of them are not as polymorphic as widely used short tandem repeat (STR) markers. In this study, 59 new highly polymorphic microhaplotypes were identified and sequenced with the NextSeq 500 Sequencer. Based on the whole 1000 Genomes Project dataset, the average effective number of alleles (Ae) of the 59 microhaplotypes was 5.44, and the Ae values of 36 of these microhaplotypes were > 5.00. Their genetic variations in 187 Han Chinese individuals were evaluated. The average allele coverage ratio (ACR) of heterozygotes across all loci was 0.96 ± 0.05. The number of observed alleles varied from 4 to 23, with an average of 8.8 alleles per microhaplotype locus. The average observed heterozygosity (Ho) of 59 loci was 0.77 ± 0.05, and the Ho values of 15 of these loci were > 0.80. All loci showed high polymorphisms with a discrimination power (DP) ranging from 0.80 to 0.97, and the average DP was 0.92 ± 0.03. The analysis of simulated mixtures demonstrated that the microhaplotypes reported here were highly polymorphic and performed well in forensic DNA mixture analysis. This study not only demonstrated the applicability of microhaplotypes in mixture analysis but also provided new choices for highly polymorphic microhaplotypes because after adding the markers identified here, the number of microhaplotypes with Ae values of > 4.00 will increase from ~ 50 to ~ 110.

Evaluation of microhaplotypes in forensic kinship analysis from a Swedish population perspective.

The development of massively parallel sequencing (MPS) technology has enabled the discovery of several new types of forensic markers where microhaplotypes are one of these promising novel genetic markers. Microhaplotypes are, commonly, less than 300 nucleotides in length and consist of two or more closely linked single-nucleotide polymorphisms (SNPs). In this study, we have examined a custom-made QIAseq Microhaplotype panel (Qiagen), including 45 different microhaplotype loci. DNA libraries were prepared according to the GeneRead DNAseq Targeted Panels V2 library preparation workflow (Qiagen) and sequenced on a MiSeq FGx instrument (Verogen). We evaluated the performance of the panel based on 75 samples of Swedish origin and haplotype frequencies were established. We performed sensitivity studies and could detect haplotypes at input amounts down to 0.8 ng. We also studied mixture samples with two contributors for which haplotypes, for the minor contributor, were detectable down to the level of 1:100. Furthermore, we executed kinship simulations to evaluate the usefulness of this panel in kinship analysis. The results showed that both paternity and full sibling cases can clearly be solved. When simulating a half sibling versus unrelated case scenario, there were, however, some overlap of the likelihood ratio distributions potentially resulting in inconclusiveness. To conclude, the results of this initial study are promising for further implementation of this microhaplotype assay into the forensic field, although we noticed some primer design issues that could be optimized, which possibly would increase the power of the assay.

Forensic genomics research on microhaplotypes.

Microhaplotype loci (microhaplotype, MHs), defined by two or more closely linked single nucleotide polymorphisms, are a type of molecular marker within a short segment of DNA. As emerging forensic genetic markers, MHs have no stutter artefacts and higher polymorphism, and permit the design of smaller amplicons. In order to identify the markers from a genome wide perspective and explore their potential application further, we constructed the most comprehensive MH dataset to date, based on the whole genome sequencing data of 105 Han individuals in Southern China from 1000 Genomes Project. The results showed that there were 9,490,075 MH loci in the range of 350 bp in the human genome, and the distribution density of microhaplotypes suggests gene variation. Polymorphism analysis of MHs from various base spans showed that the polymorphism of MHs could reach or exceed common short tandem repeat sites. In addition, based on their flexible assembly, a scheme to build the public database of microhaplotypes was proposed.

A new set of DIP-SNP markers for detection of unbalanced and degraded DNA mixtures.

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP-SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP-SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP-SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP-SNPs in a Chinese Han population. The DIP-SNPs were capable of detecting the minor contributor's allele in home-made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1 -10 ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP-SNPs may serve as a valuable tool in detection of UDM in forensic medicine.

MHBase: A comprehensive database of short microhaplotypes for advancing forensic genetic analysis.

Microhaplotypes (MHs) were first recommended by Prof. Kidd for use in forensics because they can improve human identification, kinship analysis, mixture deconvolution, and ancestry prediction. Since their introduction, extensive research has demonstrated the advantages of MHs in forensic applications and provided useful data for different populations. Currently, two databases, ALFRED (ALlele FREquency Database) and MicroHapDB (MicroHaplotype DataBase), house the published MH information and population data. We previously constructed a single nucleotide polymorphism SNP-SNP MH database (D-SNPsDB) of MHs within 50 bp on the whole human genome for 26 populations integrating basic data such as physical genome positions, mapping of variant identifiers (rsIDs), allele frequencies, and basic variant information. Building upon the previous research, we further selected MHs containing at least two variants (SNPs and/or insertions/deletions [InDels]) within a short DNA fragment (≤ 50 bp) in 26 populations based on the 1000 Genomes Project dataset (Phase 3) to construct a more comprehensive database. Subsequently, we established a user-friendly website that allows users to search the MH database (MHBase) based on their research objectives and study population to find suitable loci and provides other functions such as querying reported loci, performing online calculations using the PHASE software, and calculating ancestral-related parameters. The loci in the database are classified as SNP-based MHs, which include only SNPs, and InDel-including MHs, which contain at least one InDel. Here, we provide a detailed overview of the MHBase and an analysis of shared loci at the global and continental levels, ancestral markers, the genetic distance within loci, and mapping with the genome annotation file. The website is an accessible and useful tool for researchers engaged in marker discovery, population studies, assay development, and panel design.

Pairwise kinship inference and pedigree reconstruction using 91 microhaplotypes.

Kinship inference has been a major issue in forensic genetics, and it remains to be solved when there is no prior hypothesis and the relationships between multiple individuals are unknown. In this study, we genotyped 91 microhaplotypes from 46 pedigree samples using massive parallel sequencing and inferred their relatedness by calculating the likelihood ratio (LR). Based on simulated and real data, different treatments were applied in the presence and absence of relatedness assumptions. The pedigree of multiple individuals was reconstructed by calculating pedigree likelihoods based on real pedigree samples. The results showed that the 91 MHs could discriminate pairs of second-degree relatives from unrelated individuals. And more highly polymorphic loci were needed to discriminate the pairs of second-degree or more distant relative from other degrees of relationship, but correct classification could be obtained by expanding the suspected relationship searched to other relationships with lower LR values. Multiple individuals with unknown relationships can be successfully reconstructed if they are closely related. Our study provides a solution for kinship inference when there are no prior assumptions, and explores the possibility of pedigree reconstruction when the relationships of multiple individuals are unknown.

Performance comparison of a previously validated microhaplotype panel and a forensic STR panel for DNA mixture analysis.

Short Tandem Repeats (STRs) are the most widespread markers in forensic genetics. However, STR stutter peaks can mask alleles from a minor contributor when analysing mixtures, hindering the interpretation of complex profiles. In this study we compared the performance of a previously described panel of microhaplotypes (MHs), an alternative type of forensic marker, against a standard STR kit. The parameters evaluated included: capability of determining the minimum number of contributors in the mixture; percentages of allele drop-outs and drop-ins; retrieval of alleles belonging to the minor contributor, and estimation of likelihood ratio (LR) values. In addition, the capacity of EuroForMix software to estimate each donor's percentage of contribution was tested, as well as the impact on results when using manually, or automatically prepared libraries. The MH panel showed better performance than STRs for the detection of 2-contributor mixtures, but the lower degree of polymorphism per MH marker hindered the task of deconvolution with multiple contributors. MHs presented higher drop-in rates and lower drop-out rates, a higher capability to recover the minor contributor's alleles and provided higher LR values than STRs, likely due to the much higher number of loci combined in the panel. Estimations of contributor ratios using EuroForMix showed promising results and marginal differences were found in these values between manually and automatically prepared libraries. Overall, results showed that the mixture detection performance of the MH panel was better or equal to the standard forensic autosomal STR panel, indicating microhaplotypes are informative markers for this purpose.

Using simulated microhaplotype genotyping data to evaluate the value of machine learning algorithms for inferring DNA mixture contributor numbers.

Inferring the number of contributors (NoC) is a crucial step in interpreting DNA mixtures, as it directly affects the accuracy of the likelihood ratio calculation and the assessment of evidence strength. However, obtaining the correct NoC in complex DNA mixtures remains challenging due to the high degree of allele sharing and dropout. This study aimed to analyze the impact of allele sharing and dropout on NoC inference in complex DNA mixtures when using microhaplotypes (MH). The effectiveness and value of highly polymorphic MH for NoC inference in complex DNA mixtures were evaluated through comparing the performance of three NoC inference methods, including maximum allele count (MAC) method, maximum likelihood estimation (MLE) method, and random forest classification (RFC) algorithm. In this study, we selected the top 100 most polymorphic MH from the Southern Han Chinese (CHS) population, and simulated over 40 million complex DNA mixture profiles with the NoC ranging from 2 to 8. These profiles involve unrelated individuals (RM type) and related pairs of individuals, including parent-offspring pairs (PO type), full-sibling pairs (FS type), and second-degree kinship pairs (SE type). Our results indicated that how the number of detected alleles in DNA mixture profiles varied with the markers' polymorphism, kinship's involvement, NoC, and dropout settings. Across different types of DNA mixtures, the MAC and MLE methods performed best in the RM type, followed by SE, FS, and PO types, while RFC models showed the best performance in the PO type, followed by RM, SE, and FS types. The recall of all three methods for NoC inference were decreased as the NoC and dropout levels increased. Furthermore, the MLE method performed better at low NoC, whereas RFC models excelled at high NoC and/or high dropout levels, regardless of the availability of a priori information about related pairs of individuals in DNA mixtures. However, the RFC models which considered the aforementioned priori information and were trained specifically on each type of DNA mixture profiles, outperformed RFC_ALL model that did not consider such information. Finally, we provided recommendations for model building when applying machine learning algorithms to NoC inference.

Efficient population representation with more genetic markers increases performance of a steelhead (Oncorhynchus mykiss) genetic stock identification baseline.

Genetic stock identification (GSI) is an important fisheries management tool to identify the origin of fish harvested in mixed stock fisheries. Periodic updates of genetic baselines can improve performance via the addition of unsampled or under-sampled populations and the inclusion of more informative markers. We used a combination of baselines to evaluate how population representation, marker number, and marker type affected the performance and accuracy of genetic stock assignments (self-assignment, bias, and holdout group tests) for steelhead (Oncorhynchus mykiss) in the Snake River basin. First, we compared the performance of an existing genetic baseline with a newly developed one which had a reduced number of individuals from more populations using the same set of markers. Self-assignment rates were significantly higher (p < 0.001; +5.4%) for the older, larger baseline, bias did not differ significantly between the two, but there was a significant improvement in performance for the new baseline in holdout results (p < 0.001; mean increase of 25.0%). Second, we compared the performance of the new baseline with increased numbers of genetic markers (~2x increase of single-nucleotide polymorphisms; SNPs) for the same set of baseline individuals. In this comparison, results produced significantly higher rates of self-assignment (p < 0.001; +9.7%) but neither bias nor leave-one-out were significantly affected. Third, we compared 334 SNPs versus opportunistically discovered microhaplotypes from the same amplicons for the new baseline, and showed the latter produced significantly higher rates of self-assignment (p < 0.01; +2.6%), similar bias, but slightly lower holdout performance (-0.1%). Combined, we show the performance of genetic baselines can be improved via representative and efficient sampling, that increased marker number consistently improved performance over the original baseline, and that opportunistic discovery of microhaplotypes can lead to small improvements in GSI performance.

Evaluation of a SNP-STR haplotype panel for forensic genotype imputation.

Short tandem repeat polymorphism (STR)-based individual identification is a popular and reliable method in many forensic applications. However, STRs still frequently fail to find any matched records. In such cases, if known STRs could provide more information, it would be very helpful to solve specific problems. Genotype imputation has long been used in the study of single nucleotide polymorphisms (SNPs) and has recently been introduced into forensic fields. The idea is that, through a reference haplotype panel containing SNPs and STRs, we can obtain unknown genetic information through genotype imputation based on known STR or SNP genotypes. Several recent studies have already demonstrated this exciting idea, and a 1000 Genomes SNP-STR haplotype panel has also been released. To further study the performance of genotype imputation in forensic fields, we collected STR, microhaplotype (MH) and SNP array genotypes from Chinese Han population individuals and then performed genotype imputation analysis based on the released reference panel. As a result, the average locus imputation accuracy was ∼83 % (or ∼70 %) when SNPs in the SNP array (or MH SNPs) were imputed from STRs, and was ∼30 % when highly polymorphic markers (STRs and MHs) were imputed from each other. When STRs were imputed from SNP array, the average locus imputation accuracy increased to ∼48 %. After analyzing the match scores between real STRs and the STRs imputed from SNPs, ∼80 % of studied STR records can be connected to corresponding SNP records, which may help for individual identification. Our results indicate that genotype imputation has great potential for forensic applications.

Are rabid raccoons (Procyon lotor) ready for the rapture? Determining the geographic origin of rabies virus-infected raccoons using RADcapture and microhaplotypes.

North America is recognized for the exceptional richness of rabies virus (RV) wildlife reservoir species. Management of RV is accomplished through vaccination targeting mesocarnivore reservoir populations, such as the raccoon (Procyon lotor) in Eastern North America. Raccoons are a common generalist species, and populations may reach high densities in developed areas, which can result in contact with humans and pets with potential exposures to the raccoon variant of RV throughout the eastern United States. Understanding the spatial movement of RV by raccoon populations is important for monitoring and refining strategies supporting the landscape-level control and local elimination of this lethal zoonosis. We developed a high-throughput genotyping panel for raccoons based on hundreds of microhaplotypes to identify population structure and genetic diversity relevant to rabies management programs. Throughout the eastern United States, we identified hierarchical population genetic structure with clusters that were connected through isolation-by-distance. We also illustrate that this genotyping approach can be used to support real-time management priorities by identifying the geographic origin of a rabid raccoon that was collected in an area of the United States that had been raccoon RV-free for 8 years. The results from this study and the utility of the microhaplotype panel and genotyping method will provide managers with information on raccoon ecology that can be incorporated into future management decisions.

Adapting an established Ampliseq microhaplotype panel to nanopore sequencing through direct PCR.

We have adapted an established Ampliseq microhaplotype panel for nanopore sequencing with the Oxford Nanopore Technologies (ONT) system, as a cost-effective and highly scalable solution for forensic genetics applications. For this purpose, we designed a protocol combining direct PCR amplification from unextracted DNA with ONT library construction and sequencing using the MinION device and workflow. The analysis of reference samples at input amounts of 5-10 ng of DNA demonstrates stable coverage patterns, allele balance, and strand bias, reaching profile completeness and concordance rates of ∼95%. Similar levels were achieved when using direct-PCR from blood, buccal and semen swabs. Dilution series results indicate sensitivity is maintained down to 250 pg of input DNA, and informative profiles are produced down to 62.5 pg. Finally, we demonstrated the forensic utility of the nanopore workflow by analyzing two third degree pedigrees that showed low likelihood ratio values after the analysis of an extended panel of 38 STRs, achieving likelihood ratios 2-3 orders of magnitude higher when testing with the MinION-based haplotype data.

Massively parallel sequencing of 74 microhaplotypes and forensic characteristics in three Chinese Sino-Tibetan populations.

Microhaplotype (MH), as an emerging type of forensic genetic marker in recent years, has the potential to support multiple forensic applications, especially for mixture deconvolution and biogeographic ancestry inference. Herein, we investigated the genotype data of 74 MHs included in a novel MH panel, the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel, in three Chinese Sino-Tibetan populations (Han, Tibetan, and Yi) using the Ion Torrent semiconductor sequencing. The sequencing performance, allele frequencies, effective number of alleles (Ae), informativeness (In), and forensic parameters were subsequently estimated and calculated. In addition, principal component analysis (PCA) and structure analysis were performed to explore the population relationships among the three populations and the ancestry component distribution. Overall, this novel MH panel is robust and reliable, and has an excellent sequencing performance. The Ae values ranged from 1.0126 to 7.0855 across all samples, and 75.68 % of MHs had Ae values >2.0000. Allele frequencies at some loci varied considerably among the three studied populations, and the mean In value was 0.0195. Moreover, the genetic affinity between Tibetans and Yis was closer than that between Tibetans and Hans. The aforementioned results suggest that the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel is highly polymorphic in three investigated populations and could be used as an effective tool for human forensics. Although these 74 MHs have demonstrated the competency in continental population stratification, a higher resolution for distinguishing intracontinental subpopulations and a more comprehensive database with sufficient reference population data still remain to be accomplished.

Development and evaluation of a novel panel containing 188 microhaplotypes for 2nd-degree kinship testing in the Hebei Han population.

Distant kinship identification is one of the critical problems in forensic genetics. As a new type of genetic marker defined and discussed in the last decade, the microhaplotype (MH) has drawn much attention in such identification owing to its specific advantages to traditional short tandem repeat (STR) or single nucleotide polymorphism (SNP) markers. In this study, MH markers were screened step by step from the 1000 Genomes Project database, and a novel multiplex panel containing 188 MHs (in which 181 are reported the first time, while 1 was reported in a previous study and the other 6 have partial overlaps with known markers) was constructed for application in 2nd- and 3rd-degree kinship identification. Along with the construction, a novel MH nomenclature was proposed, in which the SNP position information they contained was taken into account to eliminate the possibility that the same locus was named differently interlaboratory. After a series of evaluations, the panel was shown to have good sequencing accuracy, high sensitivity, species specificity, and resistance to anti-PCR inhibitors or degradation. Population data of the 188 MHs were calculated based on the genetic information of 221 unrelated Hebei Han individuals, and the effective number of alleles (Ae) ranged from 2.0925 to 8.2634 (with an average of 2.9267). For the whole system, the cumulative matching probability (CMP), the cumulative power of exclusion in paternity testing of duos (CPEduo) and that of trios (CPEtrio) reached 2.8422 × 10-137, 1-1.3109 × 10-21, and 1-2.8975 × 10-39, respectively, indicating that this panel was satisfactory for individual identification and paternity testing. Then, the efficiency of the 188 MHs in 2nd- and 3rd-degree kinship testing was studied based on 30 extended families consisting of 179 2nd-degree and 121 3rd-degree relatives, as well as simulations of 0.5 million pairs of those two kinships. The results showed that clear opinions would be given in 83.36% of 2nd-degree identifications with a false rate less than 10-5, when the confirming and excluding thresholds of cumulative likelihood ratio (CLR) were set as 104 and 10-4, respectively. This panel is still not sufficient to solve the problem of 3rd-degree kinship identification alone, and approximately 300 or 870 MH loci would be needed in 2nd- or 3rd-degree kinship identification, respectively, to achieve a system efficiency not less than 0.99 with such a threshold set; such necessary numbers would be used only as a reference in further research.

Development and evaluations of the ancestry informative markers of the VISAGE Enhanced Tool for Appearance and Ancestry.

The VISAGE Enhanced Tool for Appearance and Ancestry (ET) has been designed to combine markers for the prediction of bio-geographical ancestry plus a range of externally visible characteristics into a single massively parallel sequencing (MPS) assay. We describe the development of the ancestry panel markers used in ET, and the enhanced analyses they provide compared to previous MPS-based forensic ancestry assays. As well as established autosomal single nucleotide polymorphisms (SNPs) that differentiate sub-Saharan African, European, East Asian, South Asian, Native American, and Oceanian populations, ET includes autosomal SNPs able to efficiently differentiate populations from Middle East regions. The ability of the ET autosomal ancestry SNPs to distinguish Middle East populations from other continentally defined population groups is such that characteristic patterns for this region can be discerned in genetic cluster analysis using STRUCTURE. Joint cluster membership estimates showing individual co-ancestry that signals North African or East African origins were detected, or cluster patterns were seen that indicate origins from central and Eastern regions of the Middle East. In addition to an augmented panel of autosomal SNPs, ET includes panels of 85 Y-SNPs, 16 X-SNPs and 21 autosomal Microhaplotypes. The Y- and X-SNPs provide a distinct method for obtaining extra detail about co-ancestry patterns identified in males with admixed backgrounds. This study used the 1000 Genomes admixed African and admixed American sample sets to fully explore these enhancements to the analysis of individual co-ancestry. Samples from urban and rural Brazil with contrasting distributions of African, European, and Native American co-ancestry were also studied to gauge the efficiency of combining Y- and X-SNP data for this purpose. The small panel of Microhaplotypes incorporated in ET were selected because they showed the highest levels of haplotype diversity amongst the seven population groups we sought to differentiate. Microhaplotype data was not formally combined with single-site SNP genotypes to analyse ancestry. However, the haplotype sequence reads obtained with ET from these loci creates an effective system for de-convoluting two-contributor mixed DNA. We made simple mixture experiments to demonstrate that when the contributors have different ancestries and the mixture ratios are imbalanced (i.e., not 1:1 mixtures) the ET Microhaplotype panel is an informative system to infer ancestry when this differs between the contributors.

Screening and selection of 21 novel microhaplotype markers for ancestry inference in ten Chinese subpopulations.

Genetic findings suggested ethnolinguistically diverse populations in China harbor differentiated genetic structure and complex evolutionary admixture histories, providing the genetic basis and theoretical foundation for forensic biogeographical ancestry inference (BGAI). Forensic assays for BGAI among intracontinental eastern Eurasians were previously conducted mainly based on SNPs or InDels. Microhaplotypes, as a set of closely linked SNPs within 200 base pairs, possess the advantages of both STRs and SNPs and have great potential in forensic ancestry inference. However, the forensic assay developed based on ancestry-informative microhaplotypes in the BGAI remains to be further explored, especially in China, which has rich genetic diversity. We described a new BGAI panel based on 21 novel identified ancestry-informative microhaplotypes that focused on dissected finer-scale ancestry compositions of Chinese populations. We initially screened all possible microhaplotypes with high Fst values among five East Asian populations and finally employed 21 candidate microhaplotypes in two multiplex SNaPshot assays. Forensic amplification efficiency and statistically/physically phased haplotypes of the 21 microhaplotypes were validated using SNaPshot and massively parallel sequencing (MPS) platforms. Next, we validated the efficiency of these microhaplotypes for BGAI in 764 individuals from ten Chinese populations using SNaPshot technology. The fine-scale ancestry source and ancestry proportion estimated by principal component analysis (PCA), multidimensional scaling (MDS), phylogenetic tree and model-based STRUCTURE among worldwide populations and East Asians showed that our customized panel could provide a higher discrimination resolution in both continental population stratification and East Asian regional substructure. East Asian populations could be classified into linguistically/geographically different intracontinental subpopulations (Tibeto-Burman, Tai-Kadai and others). Finally, we obtained a higher estimated accuracy using training and tested datasets in the microhaplotype-based panel than traditional SNP-based panels. Generally, the above results demonstrated that this microhaplotype panel was robust and suitable for forensic BGAI in Chinese populations, which provided high discriminatory power for continental populations and discriminated East Asians into linguistically restricted subpopulations.

Identification of missing persons through kinship analysis by microhaplotype sequencing of single-source DNA and two-person DNA mixtures.

In forensic applications, there is an increasing demand for the analysis of DNA profiles arising from missing person identification (MPI) cases. A specific DNA profile may originate from a single source or more than one contributor (i.e., a DNA mixture). When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. As a novel kind of multiallelic marker, microhaplotypes have proven promising for relatedness determination and mixture deconvolution. Herein, we developed a large panel of 185 microhaplotype markers and demonstrated its application in different scenarios of relationship inference through a simulation study and real pedigree analysis, combined with probabilistic genotyping models for data interpretation. Based on single-source profiles, it was shown that the present microhaplotype panel was sufficient for pairwise close relative testing (parent/child, full-sibling and 2nd-degree relative). For more distant relatives (3rd-degree relatives), there was a clear improvement when data from one well-chosen extra relative were available. We further sought to evaluate the theoretical systematic effectiveness and actual performance of microhaplotype markers in identifying the contribution of a missing pedigree member to a two-person mixture (as a minor donor). It was observed that 100% correct assignments were made in the balanced mixtures (with no dropout) when referenced to close relatives. When the mixture profiles suffered from dropout, incorrect assignments of minor donors were markedly associated with relatedness and the dropout level. Meanwhile, the studied scenarios generally exhibited zero or very low false-positive rates, indicating a low probability of incorrectly assigning an unrelated contributor as a close relative of the reference. Our results indicate that microhaplotype data can be reliably interpreted for identifying missing persons through kinship analysis based on DNA profiles of single-source samples or two-person mixtures. Furthermore, this study could be extended to more complex scenarios, such as determining the relatedness of contributors in (or among) mixed DNA profiles, if combined with different statistical frameworks.