Forensic Genetic Markers Research Articles

Identification of sequence polymorphisms at 58 STRs and 94 iiSNPs in a Tibetan population using massively parallel sequencing

Massively parallel sequencing (MPS) has rapidly become a promising method for forensic DNA typing, due to its ability to detect a large number of markers and samples simultaneously in a single reaction, and sequence information can be obtained directly. In the present study, two kinds of forensic genetic markers, short tandem repeat (STR) and identity-informative single nucleotide polymorphism (iiSNP) were analyzed simultaneously using ForenSeq DNA Signature Prep Kit, a commercially available kit on MPS platform. A total of 152 DNA markers, including 27 autosomal STR (A-STR) loci, 24 Y chromosomal STR (Y-STR) loci, 7 X chromosomal STR (X-STR) loci and 94 iiSNP loci were genotyped for 107 Tibetan individuals (53 males and 54 females). Compared with length-based STR typing methods, 112 more A-STR alleles, 41 more Y-STR alleles, and 24 more X-STR alleles were observed at 17 A-STRs, 9 Y-STRs, and 5 X-STRs using sequence-based approaches. Thirty-nine novel sequence variations were observed at 20 STR loci. When the flanking regions were also analyzed in addition to target SNPs at the 94 iiSNPs, 38 more alleles were identified. Our study provided an adequate genotype and frequencies data of the two types of genetic markers for forensic practice. Moreover, we also proved that this panel is highly polymorphic and informative in Tibetan population, and should be efficient in forensic kinship testing and personal identification cases.

Mutational data and population profiling of 23 Y-STRs in three Brazilian populations

Y-chromosomal STRs are important markers in forensic genetics, due to some peculiar characteristics. The absence of recombination makes them a useful tool to infer kinship in complex cases involving distant paternal relatives, or to infer paternal bio-geographic ancestry. The presence of a single copy, being transmitted from father to son, allow tracing mutational events in Y-STRs without ambiguity. For the statistical interpretation of forensic evidences based on Y-STR profiles, it is necessary to have estimates on both mutation rates and haplotype frequencies. In this work, 407 father-son duos from São Paulo and Rio de Janeiro states and 204 unrelated individuals from Manaus were analyzed. Haplotype frequencies and mutation rates for the Y-STRs from the PowerPlex Y23 commercial kit were estimated. Thirty-six mutations were observed in 15 of the 22 Y-STRs analyzed, for an average mutation rate of 3.84 × 10−3 (95 % CI 2.69 × 10−3 to 5.32 × 10−3). All mutations in GAAA repeats occurred in alleles with 13 or more uninterrupted units. Mutations in GATA repeats were observed in alleles with 9–17 uninterrupted units. An analysis carried out in different father's age groups showed an increase of 2.48 times the mutation rate in the age group of 40–50 years, when compared to the 20–30 age group, in agreement with the described for autosomal STRs. A high haplotype diversity was found in the three Brazilian populations. Pairwise genetic distance analysis (FST) showed no significant differences between the three populations in this study, which were also close to populations with strong European influence. The highest distances among the Brazilian populations were with São Gabriel da Cachoeira, which has a high Native American ancestry.

A new approach to detect a set of SNP-SNP markers: Combining ARMS-PCR with SNaPshot technology.

Microhaplotypes are a new promising type of forensic genetic marker. Without the interference of stutter and high mutation rates as for STRs, and with short amplification lengths and a higher degree of polymorphism than single SNP, microhaplotypes composed of two SNPs, SNP-SNP, have a strong application potential. Currently, the most common method to detect microhaplotypes is massive parallel sequencing. However, the cost and extensive use of instruments limit its wide application in forensic laboratories. In this study, we screened 23 new SNP-SNP loci and established a new detection method by combining a multiplex amplification refractory mutation system-based PCR (ARMS-PCR) and SNaPshot technology based on CE. First, we introduced an additional deliberate mismatch at the antepenultimate base from the 3' end of primers when designing ARMS-PCR for SNP 1 (the first SNP of the SNP-SNP). Then, single base extension primers for SNaPshot assay were designed next to the position of SNP 2 (the second SNP). Finally, 15 loci were successfully built into four panels and these loci showed a relatively high level of polymorphism in the Southwest Chinese Han population. All the loci had an average probability of informative genotypes (I value) of 0.319 and a combined discrimination power of 0.999999999. Therefore, this new detection system will provide a valuable supplement to current methods.

A 124-plex Microhaplotype Panel Based on Next-generation Sequencing Developed for Forensic Applications

Microhaplotypes are an emerging type of forensic genetic marker that are expected to support multiple forensic applications. Here, we developed a 124-plex panel for microhaplotype genotyping based on next-generation sequencing (NGS). The panel yielded intralocus and interlocus balanced sequencing data with a high percentage of effective reads. A full genotype was determined with as little as 0.1 ng of input DNA. Parallel mixture experiments and in-depth comparative analyses were performed with capillary-electrophoresis-based short tandem repeat (STR) and NGS-based microhaplotype genotyping, and demonstrated that microhaplotypes are far superior to STRs for mixture deconvolution. DNA from Han Chinese individuals (n = 256) was sequenced with the 124-plex panel. In total, 514 alleles were observed, and the forensic genetic parameters were calculated. A comparison of the forensic parameters for the 20 microhaplotypes with the top Ae values in the 124-plex panel and 20 commonly used forensic STRs showed that these microhaplotypes were as effective as STRs in identifying individuals. A linkage disequilibrium analysis showed that 106 of the 124 microhaplotypes were independently hereditary, and the combined match probability for these 106 microhaplotypes was 5.23 × 10−66. We conclude that this 124-plex microhaplotype panel is a powerful tool for forensic applications.

Recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics on forensic Y chromosome typing.

Y chromosome typing has been performed in forensic genetic practice for more than 20 years. The latest recommendations of the DNA Commission of the International Society of Forensic Genetics (ISFG) concerning the application of Y-chromosomal markers in forensic genetics were published in 2006. The aim of this report is to recapitulate, systematise and supplement existing recommendations on the forensic analysis of polymorphism of the Y chromosome with standards already implemented in practice, new capabilities linked to the development of research techniques as well as current solutions used in statistical analysis. The recommendations have been adapted specifically to aspects related to the preparation of expert opinions in the field of forensic genetics in Poland. The Polish Speaking Working Group of the ISFG believes that the presented guidelines should become a standard implemented by all Polish laboratories performing Y chromosome typing for forensic purposes.

Forensic features and genetic structure of the Hotan Uyghur inferred from 27 forensic markers

Background: The Uyghur is the fifth largest group in China and also the largest ethnic minority in Xinjiang.Aim: To explore the genetic variations of 27 forensic genetic markers included in the newly developed SureID® PanGlobal Human DNA Identification System and analyse the genetic relationship between Xinjiang Uyghur and their neighbours.Subjects and methods: We genotyped 27 markers in 2,189 unrelated Uyghur individuals from the Hotan Prefecture in Southwest Xinjiang. Comprehensive population genetic studies among Chinese populations and worldwide populations were conducted via various statistics.Results: The combined power of discrimination (CPD) and the combined power of exclusion (CPE) of the new-generation autosomal STR amplification system in the Hotan Uyghur are 9,9999-E01 and 9,9999-E01, respectively. Population genetic studies indicate that the Hotan Uyghur show a close genetic relationship with geographically different Uyghurs and Kazakhs, while significant genetic differentiation exists between the Hotan Uyghur and some ethnicities from other non-Turkic-speaking populations. The results of population comparisons among the 52 worldwide populations demonstrate that geographically approached intercontinental populations have close genetic relationships.Conclusions: 24 autosomal STRs are highly polymorphic and informative in the Uyghur and this system is suitable for forensic personal identification and paternity testing. Our findings not only reveal that Chinese Uyghur is a homogenous population based on forensic genetic markers, but also indicate that population genetic affinity is closely related to the adjacent populations with common ethnic origin.

A new proposed nomenclature for microhaplotypes

Microhaplotype marker is a new type of forensic genetic marker, and it is becoming more and more important with the development of massively parallel sequencing technology. Therefore, a unified and standardized nomenclature is required. Kenneth K. Kidd has proposed and published a nomenclature for microhaplotype markers. The nomenclature involves a root “MH” which is followed by chromosome numbers and unique characters established by the authors in their initial publication. Mh01kk001 is an example following this nomenclature system. Almost at the same time, our team proposed another similar nomenclature. After comparing it with Kidd’ nomenclature, our team improved our nomenclature and proposed a supplement for the nomenclature of microhaplotype markers. It involves five parts: the root “MH”, the chromosome number, the name of lab which published the loci initially, the individual numbers of the loci and the version number that defined by the cluster of SNPs, for example, mh01kk001A.

Performance of a massive parallel sequencing microhaplotypes assay on degraded DNA

Massively parallel sequencing (MPS) has allowed to analyze a new type of forensic genetic marker, known as microhaplotypes (MHs). MHs appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Moreover, MHs are potentially suitable for the analysis of degraded DNA samples.We designed a new panel of 29 MHs for MPS assay, with amplicons sizes below 180 bp and we investigated its effectiveness with low amounts of degraded samples. We genotyped a set of real forensic samples together with a set of artificially degraded DNAs. Also, a sensitivity test was assessed by a set of 2800 M DNA dilutions. The Depth of Coverage (DoC) were uniform across all 29 loci, in spite of amplicons size. Genotyping results shown that full profiles can be obtained even in highly degraded samples when the amount of template range from 0.1 to 5.0 ng. Finally, the increase of the number of PCR cycles did not provide an improvement in typing results of low amounts of degraded samples as, in front of higher number of typed loci, higher frequencies of artefacts leading to mistyping are found at 25 cycles.

A new approach to detect a set of SNP-SNP markers: Combining ARMS-PCR with SNaPshot technology

Microhaplotypes have become a new promising forensic genetic marker in recent years. The microhaplotype composed of two SNPs, SNP-SNP, indicates strong application potential because of the shortest fragment and good polymorphism and without the interference of stutter and high mutation rate as short tandem repeats (STR) and low polymorphism as a single SNP. Currently, the most common method to detect microhaplotypes is massively parallel sequencing (MPS), however its high cost and the need for special instruments limit its use in general forensic laboratories. In this study, we screened out 8 new SNP-SNP loci and established a new detection method by associating multiplex ARMS-PCR and SNaPshot technology. Firstly, we introduced ARMS-based PCR for SNP1. Then, SBE primers for SNaPshot assay were designed as 20–25 bp upstream complementary sequence next to the position of SNP2. Finally, 8 loci were built into one panel based on different SBE primer lengths and fluorescence colors. In brief, by combing ARMS-PCR and SNaPshot technology, it is easy and fast to profile the SNP1 and SNP2 orderly of the SNP-SNP microhaplotype based on CE platform. Our results suggested that the 8 loci have relatively high polymorphism as well as robust performance.

Development and validation of a novel SiFaSTRTM 23-plex system.

Human identification and paternity testing are usually based on the study of STRs depending on their particular characteristics in the forensic investigation. In this paper, we developed a sensitive genotyping system, SiFaSTR™ 23-plex, which is able to characterize 18 expanded Combined DNA Index System STRs (D3S1358, D5S818, D2S1338, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D10S1248, D8S1179, D1S1656, D18S51, D12S391, D19S433, D16S539, D13S317, and FGA), three highly polymorphic STRs among Chinese people (Penta D, Penta E, and D6S1043), one Y-chromosome Indel and amelogenin using a multiplex PCR; the PCR amplified products were analyzed using the Applied Biosystems 3500 Genetic Analyzer. Full genotyping profiles were obtained using only 31.25 pg of control DNA; various case-type specimens, as well as ten-year-old samples were also successfully genotyped. Additionally, in the validation studies, this multiplex was demonstrated to be human-specific and compatible with the interference of multiple PCR inhibitors. The system also enabled the detection of mixtures, and complete profiles could be obtained at the mixed ratios of 1:1, 1:3, and 3:1. The development and validation study here illustrated that the SiFaSTR™ 23-plex system is accurate, powerful, and more sensitive than many other systems. What's more, the population data in our study not only illustrated that this 23-plex typing system was straightforward and efficient but also expanded the Chinese STR database, which could facilitate the appropriate application of the 23 genetic markers in forensic genetics, especially in the Chinese population.

Population Identifiability from Forensic Genetic Markers: Ancestry Variation in Latin America

Population Identifiability from Forensic Genetic Markers: Ancestry Variation in Latin America

A microhaplotypes panel for massively parallel sequencing analysis of DNA mixtures

Massively parallel sequencing (MPS) technology enables the simultaneous analysis of different kinds of forensic genetic markers as well as the detection of the alleles with minor contributions in a highly unbalanced DNA mixture. In our previous study, we presented a novel set of microhaplotype loci and evaluated their application value in analyzing the DNA mixtures. However, several issues remain unclear, for example (i) whether the major or the minor donor in an unbalanced DNA mixture can be compared with a known suspect, (ii) whether the proportion of each contributor in a DNA mixture can be inferred, (iii) whether the captured alleles can be assigned to the major or the minor donor. To further address these issues, during the present study, we increased the number of loci in a single multiplex system to 25 (all the microhaplotypes have a length less than 50 bp). The DNA samples of 60 unrelated Han Chinese individuals, 40 artificially made DNA mixtures with different mixing proportions were analyzed with MPS by using the microhaplotypes panel. By comparing the population genetic data of the 25 microhaplotypes in 26 populations from 1000 Genome Project (1000 G), the microhaplotypes were found to have similar high Ae values in different populations. The likelihood ratios were further employed to compare the mixtures and known suspects. The major contributor could be well identified in both balanced and unbalanced samples. The minor contributor could also be identified if capturing enough loci. When we divided the microhaplotypes into different subgroups, significant correlation was found between the allele depth ratio and the gradual mixing ratios. The allele depth patterns in balanced, mildly imbalanced and severely imbalanced mixtures were clarified, and we found that certain loci could be discriminated from the contributors and they could be used for direct individual identification.

Population Genetic Diversity and Phylogenetic Characteristics for High-Altitude Adaptive Kham Tibetan Revealed by DNATyperTM 19 Amplification System.

Tibetans residing in the high-altitude inhospitable environment have undergone significant natural selection of their genetic architecture. Recently, highly mutational autosomal short tandem repeats were widely used not only in the anthropology and population genetics to investigate the genetic structure and relationships, but also in the medical genetics to explore the pathogenesis of multiple genetic diseases and in the forensic science to identify individual and parentage relatedness. However, genetic variants and forensic efficiency of DNATyperTM 19 amplification system and genetic background of Kham Tibetan remain uncharacterized. Thus, we genotyped 19 forensic genetic markers in 11,402 Kham Tibetans to gain insight into the genetic diversity of Chinese high-altitude adaptive population. Highly discriminating and polymorphic forensic measures were observed, which indicated that this new-developed DNATyper 19 PCR amplification is suitable for routine forensic identification purposes and Chinese national DNA database establishment. Pairwise genetic distances among the comprehensive population comparisons suggested that this high-altitude adaptive Kham Tibetan has genetically closer relationships with lowlanders of Tibeto-Burman-speaking populations (Chengdu Tibetan, Liangshan Tibetan, and Liangshan Yi). Genetic substructure analyses via phylogenetic reconstruction, principal component analysis, and multidimensional scaling analysis in both nationwide and worldwide contexts suggested that the genetic proximity exists along the linguistic, ethnic, and continental geographical boundary. Further studies with whole-genome sequencing of modern or archaic Kham Tibetans would be useful in reconstructing the Tibetan population history.

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.

AIM-SNPtag: A computationally efficient approach for developing ancestry-informative SNP panels

Inferring an individual's ancestry or group membership using a small set of highly informative genetic markers is very useful in forensic and medical genetics. However, given the huge amount of SNP data available from a diverse of populations, it is challenging to develop informative panels by exhaustively searching for all possible SNP combinations. In this study, we formulate it as an algorithm problem of selecting an optimal set of SNPs that maximizes the inference accuracy while minimizes the set size. Built on this conception, we develop a computational approach that is capable of constructing ancestry informative panels from multi-population genome-wide SNP data efficiently. We evaluated the performance of the method by comparing the panel size and membership inference accuracy of the constructed SNP panels to panels selected through empirical procedures in previous studies. For the membership inference of population groups including Asian, European, African, East Asian and Southeast Asian, a 36-SNP panel developed by our approach has an overall accuracy of 99.07%, and a 21-SNP subset of the panel has an overall accuracy of 95.36%. In comparison, an existing panel requires 74 SNPs to achieve an accuracy of 94.14% on the same set of population groups. We further apply the method to four subpopulations within Europe (Finnish, British, Spanish and Italian); a 175-SNP panel can discriminate individuals of those European subpopulations with an accuracy of 99.36%, of which a 68-SNP subset can achieve an accuracy of 95.07%. We expect our method to be a useful tool for constructing ancestry informative markers in forensic genetics.

Sequencing of 231 forensic genetic markers using the MiSeq FGx™ forensic genomics system – an evaluation of the assay and software

ABSTRACTThe MiSeq FGx™ Forensic Genomics System types 231 genetic markers in one multiplex polymerase chain reaction (PCR) assay. The markers include core forensic short tandem repeats (STRs) as well as identity, ancestry and phenotype informative short nucleotide polymorphisms (SNPs). In this work, the MiSeq FGx™ Forensic Genomics System was evaluated by analysing reproducibility, sensitivity, mixture identification and forensic phenotyping capabilities of the assay. Furthermore, the genotype calling of the ForenSeq™ Universal Analysis Software was verified by analysing fastq.gz files from the MiSeq FGx™ platform using the softwares STRinNGS and GATK. Overall, the performance of the MiSeq FGx™ Forensic Genomics System was high. However, locus and allele drop-outs were relatively frequent at six loci (two STRs and four human identification SNPs) due to low read depth or skewed heterozygote balances, and the stutter ratios were larger than those observed with conventional STR genotyping methods. The risk of locus and allele drop-outs increased dramatically when the amount of DNA in the first PCR was lower than 250 pg. Two-person 50:1 mixtures were identified as mixtures, whereas 100:1 and 1 000:1 mixtures were not. Y-chromosomal short tandem repeats (Y-STRs) alleles were detected in the 100:1 and 1 000:1 female/male mixtures. The ForenSeq™ Universal Analysis Software provided the data analyst with useful alerts that simplified the analysis of the large number of markers. Many of the alerts were due to user-defined, locus-specific criteria. The results shown here indicated that the default settings should be altered for some loci. Also, recommended changes to the assay and software are discussed.

Population Identifiability from Forensic Genetic Markers: Ancestry Variation in Latin America.

The Combined DNA Index System (CODIS) loci comprise a standard microsatellite marker set widely used for distinguishing among individuals in forensic DNA identity testing for medicolegal casework in the United States and in other countries. In anthropological genetic research, CODIS markers have become an important tool for uses extending beyond case investigations to quantify ancestry proportions, reveals patterns of admixture, and trace population histories. These investigations are especially prevalent in studies of Latin American population structure. Nevertheless, the accuracy of the ancestry estimates computed from the CODIS loci for highly admixed Latino populations has not been formally tested. Longstanding arguments have been made that small ancestry panels, including the CODIS loci specifically, are not suitable for ancestry inference in admixed populations, due to high heterozygosity and limited number of loci used. Recent studies on ancestry inference using the CODIS loci suggest that these do confer more information of population-level identifiability than recognized in forensic genetic scholarship and by the medicolegal community. Here, we formally test the ability of CODIS and CODIS-proxy (e.g., high-heterozygosity and individual-identifiability loci) marker panels to accurately estimate admixture proportions of individuals, including a sample of Latinos with a wide range of ancestry proportions. Using the same individuals to make direct comparisons of the outcomes, the authors produced ancestry estimates from (a) a small CODIS/CODIS-proxy locus panel and (b) a robust and validated microsatellite ancestry-informative panel. They found evidence (e.g., ρ = 0.80-0.88) that supports the use of CODIS/ CODIS-proxy loci to capture the general ancestry estimation trends of a sample. This finding is in line with results of studies using CODIS on Latin American populations: the ancestry estimations generated by CODIS present trends supported by documented population histories (e.g., colonialism and population movements) and microevolutionary events (e.g., gene flow) in Latin America. However, this study also highlights the limitations of CODIS for making individual-level inferences of ancestry: the associated estimates for an acceptable level of statistical confidence (95%) are too broad to make any nuanced inferences regarding an individual's actual ancestry composition.

The Status of the Social Fact

The Status of the Social Fact

Developing a new nonbinary SNP fluorescent multiplex detection system for forensic application in China.

Nonbinary single-nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent-labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis.

Application of next generation sequencing in forensic science [Yeni nesil dizilemenin adli bilimlerde kullanimi

Next Generation Sequencing (NGS) Technology has been emerged and started to use in genetic research since the 2000s. Until today, various methods which are faster, high throughput and low cost have been developed. NGS Technologies have been gradually developed and the quality of sequencing has been reached the high level and forensic genetics labs have moved to this technology for recent years. NGS Technologies provide new opportunities for forensic genetics research. NGS Technologies enable to analysis of many forensic genetic markers (as STRs, SNPs, mRNA) in only one experiment. Besides, NGS technologies are applicable for different practices which are essential in forensic researches. Among these, create of a DNA database, ancestry and phenotype studies, monozygotic twin studies, identification of body fluids and species, forensic microbiology studies can be considered. This review has been written to focus on NGS technologies in forensic genetics applications and to be a reference study for next forensic researchers.