Forensic identity SNPs: Characterisation of flanking region variation using massively parallel sequencing

Single nucleotide polymorphisms (SNPs) can be analysed for identity or kinship applications in forensic genetics to either provide an adjunct to traditional STR typing or as a stand-alone approach. The advent of massively parallel sequencing technology (MPS) has provided a useful opportunity to more easily deploy SNP typing in a forensic context, given the ability to simultaneously amplify a large number of markers. Furthermore, MPS also provides valuable sequence data for the targeted regions, which enables the detection of any additional variation seen in the flanking regions of amplicons. In this study we genotyped 977 samples across five UK-relevant population groups (White British, East Asian, South Asian, North-East African and West African) for 94 identity-informative SNP markers using the ForenSeq DNA Signature Prep Kit. Examination of flanking region variation allowed for the identification of 158 additional alleles across all populations studied. Here we present allele frequencies for all 94 identity-informative SNPs, both including and excluding the flanking region sequence of these markers. We also present information on the configuration of these SNPs in the ForenSeq DNA Signature Prep Kit, including performance metrics for the markers and investigation of bioinformatic and chemistry-based discordances. Overall, the inclusion of flanking region variation in the analysing workflow for these markers reduced the average combined match probability 2175 times across all populations, with a maximum reduction of 675,000-fold in the West African population. The gain due to flanking region-based discrimination increased the heterozygosity of some loci above that of some of the least useful forensic STR loci; thus demonstrating the benefit of enhanced analysis of currently targeted SNP markers for forensic applications.     

Characterization of eight Y-STR loci and haplotypes in a Chinese Han population

In this study we analyzed the eight Y-STR loci, DYS443, DYS444, DYS448, DYS453, DYS455, DYS456, DYS457 (DYS437) and DYS458, investigated haplotype distributions of these Y-STR loci in a Chinese Han population, and sequenced alleles of the eight loci for clarifying the structure. Extracted DNA was amplified by PCR and the PCR products were analyzed by non-denaturing horizontal polyacrylamide gel electrophoresis with a discontinuous buffer system. Alleles were sequenced on an ABI 3700 using a Dye Terminator Cycle sequencing kit. DYS443, DYS453, DYS455 and DYS456 were found to be simple repeat systems, while DYS444, DYS448, DYS457 (DYS437) and DYS458 were complex repeat systems. The gene diversities of DYS443, DYS444, DYS448, DYS453, DYS455, DYS456, DYS457 (DYS437) and DYS458 were 0.7742, 0.7671, 0.7453, 0.3545, 0.0549, 0.6988, 0.6148 and 0.8213, respectively. The haplotype diversity for 8 Y-STR loci was 0.9996, and the discrimination capacity was 0.9815. The results indicate that these eight loci are useful Y-linked markers for forensic applications.     

Forensic molecular biomarkers for mixture analysis

The deconvolution of DNA mixtures has gathered the attention of forensic DNA scientists for over two decades. To enhance mixture deconvolution capabilities, a new generation of sensitive DNA-typing approaches has been recently proposed. In this review, we describe novel, forensically relevant multi-SNP loci (i.e., microhaplotypes or microhaps), compound markers (i.e., DIP-STRs, SNP-STRs and DIP-SNPs) and lineage markers (i.e., rapidly mutating Y chromosome STRs) that improve the deconvolution of two and more than two-person mixtures typed using conventional STR, binary and non-binary loci. We explore the features and applications of these emerging molecular biomarkers with respect to their ability to forensically detect same-or-opposite sex donors. Finally, we discuss the impact of initial massively parallel sequencing (MPS) investigations of STR, microhaplotype and SNP/indel assays for DNA mixture profiling.     

Screening of highly discriminative microhaplotype markers for individual identification and mixture deconvolution in East Asian populations

Microhaplotypes are forensic genetic markers that combine single nucleotide polymorphisms in close proximity to one another. Highly discriminative microhaplotype markers could be superior to short tandem repeats (STRs) in DNA mixture deconvolution investigations because they are not interfered by stutters. In this study, the effective number of alleles (A_e) and discrimination power values of microhaplotypes and STRs were compared. It was found that current microhaplotypes are not as discriminative as commonly used forensic STRs. Effective screening of highly discriminative microhaplotype markers were consequently conducted for East Asian populations. To satisfy different forensic application needs, four sets of microhaplotypes with A_e values ≥ 4 were screened for under different conditions that included marker length and physical distances between markers. While the four sets contained 703, 301, 337, and 190 microhaplotypes, their average A_e values reached 5.38, 6.30, 7.39, and 5.61, respectively. The microhaplotype group containing 301 markers (maximum length of 200 bp and separated by ≥ 5 million bases) was further investigated. The results showed that none of the 301 loci were exactly the same as those previously reported, while seven loci partially overlapped with known markers. While A_e values of 45 loci were ≥ 8, the A_e value of the mh17WL-008 locus reached a maximum of 93.57. Further analysis showed that the newly identified microhaplotype markers were also highly polymorphic in African, American, European, and South Asian populations.     

Forensic nanopore sequencing of microhaplotype markers using QitanTech’s QNome

In recent years, extraordinary progress has been made in genome sequencing technologies, which has led to a decrease in cost and an increase in the diversity of sequenced genomes. Nanopore sequencing is one of the latest genome sequencing technologies. It aims to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions, and provides a new approach for forensic genetics to detect longer markers in real time. To date, multiple studies have been conducted to sequence forensic markers using MinION from Oxford Nanopore Technologies (ONT), and the results indicate that nanopore sequencing holds promise for forensic applications. Qitan Technology (QitanTech) recently launched its first commercial nanopore genome sequencer, QNome. It could achieve a read length of more than 150 kbp, and could generate approximately 500 Mb of data in 8 h. In this pilot study, we explored and validated this alternative nanopore sequencing device for microhaplotype (MH) profiling using a custom set of 15 MH loci. Seventy single-contributor samples were divided into 7 batches, each of which included 10 samples and control DNA 9947A and was sequenced by QNome. MH genotypes generated from QNome were compared to those from Ion Torrent sequencing (Ion S5XL system) to evaluate the accuracy and stability. Twelve samples randomly selected from the last three batches and Control DNA 9947A were also subjected to ONT MinION sequencing (with R9.4 flow cell) for parallel comparison. Based on MHtyper, a bioinformatics workflow developed for automated MH designation, all MH loci can be genotyped and reliably phased using the QNome data, with an overall accuracy of 99.83% (4 errors among 2310 genotypes). Three occurred near or in the region of homopolymer sequences, and one existed within 50 bp of the start of the sequencing reaction. In the last 15 samples (12 individual samples and 3 replicates of control DNA 9947A), two SNPs located at 4-mer homopolymers failed to obtain reliable genotypes on the MinION data. This study shows the potential of state-of-the-art nanopore sequencing methods to analyze forensic MH markers. Given the rapid pace of change, sporadic and nonrepetitive errors presented in this study are expected to be resolved by further developments of nanopore technologies and analysis tools.     

Development of a five ChX STRs loci typing system

We investigated the polymorphism of five X-chromosomal short tandem repeat markers (ChrX STRs) loci (DXS7132, DXS981, DXS6803, DXS6809, and DXS6789) and their value for forensic applications. A fluorescent multiplex polymerase chain reaction (PCR) for amplifying five ChrX STRs loci in the same PCR reaction was set up. A total of 827 unrelated individuals of the Han nationality in China were tested. The results show that the five loci in the multiplex system provide high polymorphism information for forensic identification and paternity testing, particularly for difficult paternity-deficient cases.     

A multipurpose panel of microhaplotypes for use with STR markers in casework

A small panel of highly informative loci that can be genotyped on the same equipment as the standard CODIS short tandem repeat (STR) markers has strong potential for application in forensic casework. Single nucleotide polymorphisms (SNPs) can be typed by a couple of methods on capillary electrophoresis (CE) machines and on sequencers, but the amount of information relative to the laboratory effort has hindered use of SNPs in actual casework. Insertion-deletion markers (InDels) suffer from similar problems. Microhaplotypes (MHs) are much more informative per locus but have similar technical difficulties unless they are typed by massively parallel sequencing (MPS). As forensic labs are acquiring sequencing machines, MHs become more likely to be used in casework, especially if multiplexed with STRs. Here we present the details of a multipurpose panel of 24 MHs with the highest effective number of alleles (A_e) from previous work. An augmented STR panel of 24 loci (20 CODIS markers plus four commonly typed STRs) is also considered. The A_e and ancestry informativeness (I_n) distributions of these two datasets are compared. The MH panel is shown to have better individualization and population distinction than the augmented CODIS STRs. We note that the 24 MHs should be better for mixture analyses than the STRs. Finally, we suggest that a commercial kit including both the standard CODIS markers and this set of 24 MH would greatly improve the discrimination power over that of current commercial assays.     

STRait Razor: A length-based forensic STR allele-calling tool for use with second generation sequencing data

Recent studies have demonstrated the capability of second generation sequencing (SGS) to provide coverage of short tandem repeats (STRs) found within the human genome. However, there are relatively few bioinformatic software packages capable of detecting these markers in the raw sequence data. The extant STR-calling tools are sophisticated, but are not always applicable to the analysis of the STR loci commonly used in forensic analyses. STRait Razor is a newly developed Perl-based software tool that runs on the Linux/Unix operating system and is designed to detect forensically-relevant STR alleles in FASTQ sequence data, based on allelic length. It is capable of analyzing STR loci with repeat motifs ranging from simple to complex without the need for extensive allelic sequence data. STRait Razor is designed to interpret both single-end and paired-end data and relies on intelligent parallel processing to reduce analysis time. Users are presented with a number of customization options, including variable mismatch detection parameters, as well as the ability to easily allow for the detection of alleles at new loci. In its current state, the software detects alleles for 44 autosomal and Y-chromosome STR loci. The study described herein demonstrates that STRait Razor is capable of detecting STR alleles in data generated by multiple library preparation methods and two Illumina^® sequencing instruments, with 100% concordance. The data also reveal noteworthy concepts related to the effect of different preparation chemistries and sequencing parameters on the bioinformatic detection of STR alleles.     

Evaluating 130 microhaplotypes across a global set of 83 populations

Today the primary DNA markers used in forensics are short tandem repeat (STR) polymorphisms (STRPs), initially selected because they are highly polymorphic. However, the increasingly common need to deal with samples with a mixture of DNA from two or more individuals sometimes is complicated by the inherent stutter involved with PCR amplification, especially in strongly unbalanced mixtures when the minor component coincides with the stutter range of the major component. Also, the STRPs in use provide little evidence of ancestry of a single source sample beyond broad “continental” resolution. Methodologies for analyzing DNA have become much more powerful in recent years. Massively parallel sequencing (MPS) is a new method being considered for routine use in forensics. Primarily to aid in mixture deconvolution and avoid the issue of stutter, we have begun to investigate a new type of forensic marker, microhaplotype loci, that will provide useful information on mixtures of DNA and on ancestry when typed using massively parallel sequencing (MPS). We have identified 130 loci and estimated their haplotype (allele) frequencies in 83 different population samples. Many of these loci are shown to be highly informative for individual identification and for mixture identification and deconvolution.     

Comprehensive annotated STR physical map of the human Y chromosome: Forensic implications

A plethora of Y-STR markers from diverse sources have been deposited in public databases and represent potential candidates for incorporation into the next generation of Y-STR multiplexes for forensic use. Here, based upon all of the Y-STR loci that have been deposited in the human genome database (>400), we have sequentially positioned each one along the Y chromosome using the most current human genome sequencing data (NCBI Build 35). The information derived from this work defines the number and relative position of all potentially forensically relevant Y-STR loci, their location within the physical linkage map of the Y chromosome and their relationship to structural genes. We conclude that there exists at present at least 417 separate Y-STR markers available for potential forensic use, although many of these will be found to be unsuitable for other reasons. However, from this data, we were able to identify 28 pairs of duplicated loci that were given separate DYS designations and four pairs of loci with overlapping flanking regions. Removing one locus from each set of duplicates reduced the number of potentially useful loci from 417 to 389. The derived information should be useful for workers who are designing novel Y-STR multiplexes to ensure the presence of non-synonymous loci and, if so desired, to avoid loci that lie within structural genes. It may also be useful for forensic casework practitioners (or molecular anthropologists) to aid in distinguishing between chromosomal rearrangements (such as duplications and deletions) and bona fide DNA admixtures or null alleles caused by primer binding site mutations. We illustrate the practical usefulness of the chromosomal positioning data in the design of eight multiplex systems using 94 Y-STR loci.     

Genotyping polymorphic microhaplotype markers through the Illumina® MiSeq platform for forensics

Microhaplotype markers are emerging forensic genetic markers that have received broad attention in forensics and may supplement existing genetic marker panels. Short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs) are the general genetic markers at present. Stutter and the high mutation rate of STR markers and the low polymorphism of SNP markers obstruct the solving of certain cases. Kidd proposed microhaplotype markers that encompass 2–4 SNPs. In this study, we screened microhaplotype loci through three criteria, and chose the Illumina^® MiSeq platform to sequence the new markers. A new nomenclature was proposed and Perl-based tool FLfinder was designed to genotype the microhaplotype marker. After counting the number of haplotypes in samples that were sequenced and calculating common forensic parameters, 13 loci with high polymorphism were reported. Twelve of the 13 loci had an average allele coverage ratio (ACR) of 0.72 to 0.92. Structure analysis showed that 2504 samples (1000 genome project) could be divided into 5 groupings of populations, and each one representing a continental origin. The finding indicates that microhaplotype markers could be used for individual identification and ancestry inference, and a new choice is provided for forensic practice in the future.     

Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics

SNPs that are molecularly very close (<10 kb) will generally have extremely low recombination rates, much less than 10⁻⁴. Multiple haplotypes will often exist because of the history of the origins of the variants at the different sites, rare recombinants, and the vagaries of random genetic drift and/or selection. Such multiallelic haplotype loci are potentially important in forensic work for individual identification, for defining ancestry, and for identifying familial relationships. The new DNA sequencing capabilities currently available make possible continuous runs of a few hundred base pairs so that we can now determine the allelic combination of multiple SNPs on each chromosome of an individual, i.e., the phase, for multiple SNPs within a small segment of DNA. Therefore, we have begun to identify regions, encompassing two to four SNPs with an extent of <200 bp that define multiallelic haplotype loci. We have identified candidate regions and have collected pilot data on many candidate microhaplotype loci. Here we present 31 microhaplotype loci that have at least three alleles, have high heterozygosity, are globally informative, and are statistically independent at the population level. This study of microhaplotype loci (microhaps) provides proof of principle that such markers exist and validates their usefulness for ancestry inference, lineage–clan–family inference, and individual identification. The true value of microhaplotypes will come with sequencing methods that can establish alleles unambiguously, including disentangling of mixtures, because a single sequencing run on a single strand of DNA will encompass all of the SNPs.     

Evaluation of a microhaplotypes panel for forensic genetics using massive parallel sequencing technology

Massive parallel DNA sequencing (MPS) makes it possible to explore a new type of genetic marker, known as microhaplotypes or microhaps. These loci were recently introduced in the landscape of forensic genetic and appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Microhaplotypes loci, based on 89 loci in ALFRED, were selected and their genetic variations in 100 Italian individuals were evaluated by using MPS, in order to make inference about utility of a set of microhaps in forensic genetics. After MPS, the panel was reduced to 87 microhaps, comprised of 266 different SNPs and spread across 22 human autosomes. Genotype and haplotype frequencies were estimated, as well as the effective number of alleles at each locus (A_e), which relates to the usefulness of the locus in resolution of relationships and deconvolution of DNA mixtures. Overall, the A_e values for the 87 microhaps range from 1.010 to 8.344, with about 80% showing values greater than 2.0. Noteworthy, 32 microhaps display A_e values greater than 3.0 and 18 loci A_e above 4.0.To explore the suitability of microhaplotypes in mixture deconvolution, the probability of detecting a mixture, as a function of A_e, was inferred for different groups of loci. Considering the fourteen loci with A_e between 3.0 and 3.999 the probability of detecting a mixture was at least 0.99973, while considering the ten loci with Ae between 4.0 and 4.999 the probability was at least 0.99998. Moreover, when considering just the six loci with A_e between 5.0 and 5.999 the probability of detecting a mixture was at least 0.99984, while when considering just the two loci with A_e above 6 the probability was 0.97228. Combining these 32 MH loci, the theoretical probability of detecting a mixture was 0.999999999999973. These results make the subset of 32 loci with A_e above three informative for mixture resolution.The individual matching probabilities (PI) of the 87 microhaps ranged from 0.032 to 0.9802. Considering the 32 microhap loci with A_e greater than 3.0, the cumulative PI value was 1.6 × 10⁻³³, while considering the 18 microhap loci with A_e above 4.0, the cumulative PI value was 2.34 × 10^-21. Overall the results of this study confirmed the utility of microhaps in forensic genetics.     

Forensic tri-allelic SNP genotyping using nanopore sequencing

The potential and current state-of-the-art of forensic SNP genotyping using nanopore sequencing was investigated with a panel of 16 tri-allelic single nucleotide polymorphisms (SNPs), multiplexing five samples per sequencing run. The sample set consisted of three single-source human genomic reference control DNA samples and two GEDNAP samples, simulating casework samples. The primers for the multiplex SNP-loci PCR were taken from a study which researched their value in a forensic setting using conventional single-base extension technology. Workflows for multiplexed Oxford Nanopore Technologies' 1D and 1D² sequencing were developed that provide correct genotyping of most SNP loci. Loci that are problematic for nanopore sequencing were characterized. When such loci are avoided, nanopore sequencing of forensic tri-allelic SNPs is technically feasible.     

Population genetic data of 4 multicopy Y-STR markers in Chinese

Novel Y chromosomal STR (Y-STR) markers have been continuously discovered during the past decades, promoting the widely application of Y-STRs in the area of forensic science. Here, four multicopy Y-STR markers were focused, including DYF383S1, DYF409S1, DYF411S1 and DYF371, which are rarely reported in China and differ in the number of copies on Y chromosome. Characterization of the markers was performed in population of Hunan province, China, based on sequence analysis. Allele nomenclature and allelic ladder were then developed to avoid the disunity of typing standard. To evaluate their forensic performance, gene diversity of the four loci was investigated in 548 unrelated male individuals from Hunan population. The number of haplotype was analyzed by both conservative (C-type) and expanded approach (E-type) for markers containing more than 2 copies. As detected, there were 7, 9, 13 alleles and 15, 22, 23 haplotypes for DYF383S1, DYF409S1 and DYF411S1, respectively. Thirty-two C-types and 56 E-types were found in DYF371, indicating the highest haplotype diversity (HD) among all tested loci (0.871 and 0.888 for C-type and E-type, respectively). Two other Y-STRs (DYF409S1, DYF411S1) also showed high haplotype diversity (>0.8) in the population. Combining the four loci, discrimination capacity reached 0.505 (C-type) or 0.533 (E-type), and the total HD values exceeded 0.991. The results inferred great potential of the multicopy markers to improve the resolution of paternal identification in China population.     

Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens

Though the utility of next-generation sequencing (NGS) technologies for forensic short tandem repeat (STR) typing has been evident for several years, commercially available assays and software solutions developed specifically to meet forensic needs have only recently become available. One of these, the ForenSeq™ DNA Signature Prep Kit (Illumina, Inc.) sequences 27 autosomal STR (aSTR) and 24 Y chromosome STR (Y-STR) loci (concurrent with additional nuclear markers) per multiplexed sample, with automated secondary and tertiary analyses of the data accomplished via the associated ForenSeq™ Universal Analysis Software (UAS). In this study we investigated the performance of the ForenSeq system for aSTR and Y-STR typing by examination of 151 sample libraries developed from high quality DNAs amplified at the target 1 ng template. Utilizing PCR Primer Mix B, greater than 99.5% of aSTR loci and 97.0% of Y-STR loci were recovered when 42 or fewer sample libraries were pooled for sequencing. A direct comparison of UAS developed fragment length results to capillary electrophoresis (CE) based data identified only two allele call discrepancies when no UAS quality flag was triggered. Review of the ForenSeq data indicated that most samples with total sequence read counts exceeding 40,000 could be interpreted to develop nearly complete aSTR genotypes or Y-STR haplotypes. However, markers D22S1045 and DYS392 produced poor or inconsistent results even when sample read counts were greater than 85,000. Excluding these two loci, analyst-interpreted aSTR and Y-STR ForenSeq profiles were 99.96% and 100% concordant, respectively, with CE data. In addition to demonstrating concordance on par with other CE kit to kit comparisons, the results from this study will assist laboratories seeking to develop workflows for high volume processing and analysis of aSTRs and Y-STRs from reference-type specimens using the ForenSeq system.     

Accurate profiling of forensic autosomal STRs using the Oxford Nanopore Technologies MinION device

The high variability characteristic of short tandem repeat (STR) markers is harnessed for human identification in forensic genetic analyses. Despite the power and reliability of current typing techniques, sequence-level information both within and around STRs are masked in the length-based profiles generated. Forensic STR typing using next generation sequencing (NGS) has therefore gained attention as an alternative to traditional capillary electrophoresis (CE) approaches. In this proof-of-principle study, we evaluate the forensic applicability of the newest and smallest NGS platform available – the Oxford Nanopore Technologies (ONT) MinION device. Although nanopore sequencing on the handheld MinION offers numerous advantages, including low startup cost and on-site sample processing, the relatively high error rate and lack of forensic-specific analysis software has prevented accurate profiling across STR panels in previous studies. Here we present STRspy, a streamlined method capable of producing length- and sequence-based STR allele designations from noisy, error-prone third generation sequencing reads. To assess the capabilities of STRspy, seven reference samples (female: n = 2; male: n = 5) were amplified at 15 and 30 PCR cycles using the Promega PowerSeq 46GY System and sequenced on the ONT MinION device in triplicate. Basecalled reads were then processed with STRspy using a custom database containing alleles reported in the STRSeq BioProject NIST 1036 dataset. Resultant STR allele designations and flanking region single nucleotide polymorphism (SNP) calls were compared to the manufacturer-validated genotypes for each sample. STRspy generated robust and reliable genotypes across all autosomal STR loci amplified with 30 PCR cycles, achieving 100% concordance based on both length and sequence. Furthermore, we were able to identify flanking region SNPs in the 15-cycle dataset with > 90% accuracy. These results demonstrate that when analyzed with STRspy ONT reads can reveal additional variation in and around STR loci depending on read coverage. As the first and only third generation sequencing platform-specific method to successfully profile the entire panel of autosomal STRs amplified by a commercially available multiplex, STRspy significantly increases the feasibility of nanopore sequencing in forensic applications.     

Next generation sequencing and its applications in forensic genetics

It has been almost a decade since the first next generation sequencing (NGS) technologies emerged and quickly changed the way genetic research is conducted. Today, full genomes are mapped and published almost weekly and with ever increasing speed and decreasing costs. NGS methods and platforms have matured during the last 10 years, and the quality of the sequences has reached a level where NGS is used in clinical diagnostics of humans. Forensic genetic laboratories have also explored NGS technologies and especially in the last year, there has been a small explosion in the number of scientific articles and presentations at conferences with forensic aspects of NGS. These contributions have demonstrated that NGS offers new possibilities for forensic genetic case work. More information may be obtained from unique samples in a single experiment by analyzing combinations of markers (STRs, SNPs, insertion/deletions, mRNA) that cannot be analyzed simultaneously with the standard PCR-CE methods used today. The true variation in core forensic STR loci has been uncovered, and previously unknown STR alleles have been discovered. The detailed sequence information may aid mixture interpretation and will increase the statistical weight of the evidence. In this review, we will give an introduction to NGS and single-molecule sequencing, and we will discuss the possible applications of NGS in forensic genetics.     

羊微卫星标记的研究进展

微卫星DNA在染色体上随机分布．是十分有效的遗传标记。目前在羊中应用的微卫星位点除了少数是直接从DNA文库中筛选的以外．大部分都是借用牛的微卫星住点。这些位点已被广泛的应用于父权的鉴定、遗传多样性和种群结构的分析、QTL定位、遗传图谱的构建等方面的研究。然而微卫DNA的数量目前还远不能满足研究的需要。将来要分离更多的微卫星住点．构建微卫星住点的遗传和物理图谱。这样微卫星DNA才能在羊的研究中发挥更大的作用。     

Evaluation of the MHSeqTyper47 kit for forensically challenging DNA samples

Microhaplotypes have been highly regarded for forensic mixture DNA deconvolution because they do not experience interference from stutters in the same way as short tandem repeat markers, and they tend to be more polymorphic than single nucleotide polymorphism markers. However, forensic microhaplotype kits have not been reported. The MHSeqTyper47 kit genotypes 47 microhaplotype loci. In this study, MiSeq FGx sequencing metrics for MHSeqTyper47 were presented, and the genotyping accuracy of this kit was examined. The sensitivity of MHSeqTyper47 reached 62.5 pg, and full genotyping results were obtained from degraded DNA samples with degradation indexes ≤ 3.00. Full genotypes were obtained in the presence of 100 ng/μL tannin, 50 μM heme, 25 ng/μL humic acid, and 1.25 μg/μL indigo dye. In DNA mixture studies, a minimum of 31 loci of the minor contributor were correctly genotyped at 1:99 or 99:1 mixing ratios, with the cumulative random matching probability of these loci reaching 4.54 × 10⁻²⁵. Mixing ratios could be reliably predicted from two-donor DNA mixtures based on the loci with four called alleles. Taken together, these data showed that the MHSeqTyper47 kit was effective for forensically challenging DNA analysis.