Use of multi‐InDels as novel markers to analyze 13 X‐chromosome haplotype loci for forensic purposes

Many studies have been proposed to identify insertion/deletion (InDel) polymorphisms in humans for forensic genetic studies. However, the discriminatory power of InDels is limited by the poor polymorphisms of diallelic markers. To improve their discriminatory power, we developed multi‐InDel, a novel autosomal marker comprising more than two InDel loci that are tightly linked by their physical position and combined into a specific marker by a pair of PCR primers. This strategy gives at least three haplotypes for each multi‐InDel marker. Such markers can be potentially very useful in forensic applications. In this study, we focused on multi‐InDel markers located on X chromosome (ChrX). A multiplex system with 13 multi‐InDel markers, including 28 InDel loci in ChrX, was developed. To validate the multi‐InDel panel, the haplotype distribution in a population sample and in a set of pedigrees was investigated. This study demonstrates usefulness of these markers for individual identification and relationship studies. We highlight the fact that the multi‐InDel markers located on ChrX can provide new supporting information for complex kinship testing.     

A new set of 20 Multi-InDel markers for forensic application

Insertion/deletion markers (InDels) become an important marker for forensic medicine because of their compatible typing techniques with STRs and lower mutation rates. Recent years, a new kind of DNA marker named Multi-InDel was reported as characterized by two or more tightly linked InDel loci within a short length of physical position, usually 200–300 nucleotides. Many pieces of research showed that Multi-InDels had excellent application values in ancestry inference and forensic medicine. Since the identical number of insertion/deletion nucleotides of the InDel markers that composing the Multi-InDel marker, the genotypes of most reported Multi-InDels could not be directly typed by capillary electrophoresis (CE) due to the lack of length discrepancy among the composing InDel sequence. In this study, we applied a typing system of 20 Multi-InDels including 41 InDels, whose genotypes could be deduced by CE and assessed their potential applications in forensic medicine. A total of 200 unrelated Chinese Han individuals and five mother-child-father trios with proven paternity with one STR locus transmission incompatibilities from Shanxi province were genotyped by the multiplex system. The results showed that a total of 70 specific alleles were observed, more than three alleles were observed in 19 loci and seven alleles were observed in one locus. The combined probability of exclusion and the combined power of discrimination were 0.992 and 0.99999999993, respectively. This study demonstrates their potential usefulness for individual identification and paternity tests. The development of Multi-InDels provided another genetic tool inherent in higher polymorphic and lower mutation rates.     

Forensic efficacy evaluation and genetic structure exploration of the Yunnan Miao group by a multiplex InDel panel

The aim of the study was to better understand the genetic characteristics of the Miao group in China. Herein, genetic characteristics and forensic application values of 57 autosomal insertion–deletion (InDel) loci were investigated in 210 unrelated healthy individuals from the Chinese Yunnan Miao (YM) group. Meanwhile, the genetic differences in these InDels were compared among the YM group and 26 reference populations. The results of forensic statistical analyses showed that all 57 autosomal InDels were in accordance with the Hardy–Weinberg and linkage equilibria of pairwise loci in the Chinese YM group. Moreover, the combined probability of discrimination and probability of exclusion in the YM group were 0.9999999999999999999999801 and 0.999928, respectively, which indicated that the multiplex amplification including 57 autosomal InDels was suitable for forensic individual identification and paternity testing in the Chinese YM group. In addition, the results of allelic frequency distribution differential analyses, principal component analyses, phylogenetic tree reconstruction, and genetic structure analyses between the Chinese YM group and 26 reference populations revealed that the genetic similarities between the YM group and East Asian populations were more than that between the YM group and other geographical populations. This 57 autosomal InDels system can also effectively distinguish East Asian, European, and African populations.     

A novel method for the analysis of 20 multi‐Indel polymorphisms and its forensic application

Insertion/deletion polymorphisms (Indels) have been considered as potential markers for forensic DNA analysis. However, the discrimination power of Indels is relatively lower due to the poor polymorphisms of diallelic markers. Here, two to three Indel loci that were very tightly linked in physical position were combined into a specific multi‐Indel marker to improve the discrimination, as well as a multiplex that consisted of a set of multi‐Indel markers was developed for forensic purpose. Finally, a multiplex system with 20 multi‐Indel markers including 43 Indel loci from dbSNP database was constructed and DNA sample can be analyzed by this multiplex in one PCR reaction and one CE run. A total of 150 unrelated individuals from Hunan province in South‐central China were genotyped by the multiplex system. The result showed that a total of 63 specific amplicons were detected, three alleles were observed in multi‐Indel markers including two Indel loci and four alleles were observed in the markers including three Indel loci. The cumulative probability of exclusion and the accumulated discrimination power were 0.9989 and 0.9999999999994, respectively. Our result demonstrated that the strategy could be efficient to develop higher polymorphic multi‐Indel markers, and the new multiplex could provide Supporting Information for forensic application.     

Development a multiplex panel of AISNPs,multi-allelic InDels,microhaplotypes, and Y-SNP/InDel loci for multiple forensic purposes via the NGS

Recently, next generation sequencing has shown the promising application value in forensic research. In this study, we constructed a multiplex amplification system of different molecular genetic markers based on the previous selected ancestry informative single nucleotide polymorphisms (SNPs), multi-allelic insertion/deletion (InDel) polymorphisms, microhaplotypes, and Y-chromosomal SNP/InDel loci, and evaluated forensic efficiencies of the system in Chinese Shaanxi Han, Chinese Hui, and Chinese Mongolian groups via the next generation sequencing platform. Ancestry information analyses of Shaanxi Han, Hui, and Mongolian groups revealed that most Mongolian individuals could be differentiated from Shaanxi Hans and Huis based on the selected ancestry informative SNPs. Multi-allelic InDels and microhaplotypes showed the multiple allele variations and possessed relatively high genetic polymorphisms in these three groups, indicating these loci could provide higher forensic efficiencies for individual identification and paternity testing. Based on Y-chromosomal SNPs, different haplogroup distributions were observed among Shaanxi Han, Hui, and Mongolian groups. In conclusion, the self-developed system could be used to simultaneously carry out the individual identification, paternity analysis, mixture deconvolution, forensic ancestry information analysis, and Y-chromosomal haplogroup inference, which could provide more valuable investigative clues in forensic practices.     

Development of a novel multiplex polymerase chain reaction system for forensic individual identification using insertion/deletion polymorphisms

Insertion/deletion (InDel) polymorphisms have been widely used in the fields of population genetics, genetic map constructions, and forensic investigations owing to the advantages of their low mutation rates, widespread distributions in the human genome, and small amplicon sizes. In order to provide more InDels with high discrimination power in Chinese populations, we selected and constructed one novel multiplex PCR‐InDel panel for forensic individual identification. Genetic distributions of these 35 InDels in five reference populations from East Asia showed low genetic differentiations among these populations. Forensic efficiency evaluations of these InDels revealed that these loci could perform well for forensic individual identifications in these reference populations. In the meantime, genetic diversities and forensic parameters of these InDels were further investigated in the studied Kazak group. Mean value of polymorphism information content for 35 InDels was 0.3611. Cumulative power of discrimination of 35 InDels was 0.99999999999999603 in Kazak group. Given these results, the panel is suitable for individual identifications in the studied Kazak and these reference populations.     

Multiplex pyrosequencing of InDel markers for forensic DNA analysis

The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator^® DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator^® DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof‐of‐principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost‐effective alternative for some applications.     

Development and validation of a forensic multiplex InDel assay: The AGCU InDel 60 kit

Marker sets based on insertion/deletion polymorphisms (InDels) combine the characteristics of both short tandem repeats (STRs) and single nucleotide polymorphisms and have served as effective complementary or stand-alone systems for human identification in forensics. We developed a novel multiplex amplification detection system, designated the AGCU InDel 60 kit, containing 57 autosomal InDels, 2 Y-chromosomal InDels, and the amelogenin locus and validated the kit in a series of studies, which included tests of the PCR conditions; tests for sensitivity, species specificity, reproducibility, stability, and mock case samples; degradation studies; and a population study. The results indicated that the AGCU InDel 60 kit was accurate, specific, reproducible, stable, and robust. Complete DNA profiles were obtained even with 125 pg of human DNA. In tests of artificially degraded samples, we found that the number of alleles detected by the validated kit was considerably greater than that detected by the STR-based AGCU 21+1 kit, even as the degree of degradation increased. Additionally, 564 unrelated individuals from three Han groups were investigated using this novel system, and the values of combined power of discrimination and combined power of exclusion were not less than 1–4.9026 × 10⁻²⁴ and 1–3.1123 × 10⁻⁵, respectively. Thus, the results indicated that the novel kit was more powerful than the previous version of the InDel kit (the AGCU InDel 50 kit). Our results suggest that the AGCU InDel 60 kit can serve as an efficient tool for human forensics and a supplementary kit for population genetics research.     

Development of a new 25plex STRs typing system for forensic application

We have developed a novel STR 25‐plex florescence multiplex‐STR kit (DNATyper25) to genotype 23 autosomal and two sex‐linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non‐CODIS. The sex‐linked markers include a Y‐STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non‐human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/μL and hematin concentration <400 μM. For forensic evaluation, the 23 autosomal STRs followed the Hardy–Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10^?22, TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.     

Design and development of novel single multiplex system incorporating 26 rapidly mutating Y-STRs; 26 RM Yplex

This article details the development of a single multiplex system amplifying 26 rapidly mutating Y-STR markers. A sequenced allelic ladder, constructed for calling alleles of all loci, is introduced. The multiplex system shows the ability to address the limitations of Y-STRs commercial kits in differentiating closely related males. The multiplex performed well in the prevalidation tests and showed great potential to be used in forensic casework.     

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.     

Population genetics and forensic efficiency of twenty‐one novel microsatellite loci of Chinese Yi ethnic group

In this study, we investigated polymorphic distributions of allelic frequencies and forensic genetic parameters of 21 novel autosomal microsatellite loci from 110 unrelated healthy individuals of Chinese Yi ethnic group. Expected heterozygosity, power of discrimination, and polymorphic information content ranged from 0.617 to 0.812, 0.777 to 0.936 and 0.560 to 0.790. The microsatellite loci showed high forensic efficiency. The total discrimination power and cumulate probability of exclusion were 0.99999999999999999986902 and 0.999998818, respectively. Locus‐by‐locus allelic frequencies were compared using analysis of molecular variance (AMOVA) method, and the statistically significant differences were observed between Yi group and Russian, Tujia, Kazak, Bai, Ningxia Han, Salar, Tibetan, and Uigur groups at 5, 6, 7, 7, 7, 8, 12, and 13 loci, respectively. The results of genetic distance comparisons, genetic structure analyses, and principal component analysis all indicated that the Yi group showed relatively short genetic relationships with Russian, Salar, and Bai group. The experimental results showed that the 21 loci in the multiplex system provided highly polymorphic information and forensic efficiency for forensic individual identification and paternity testing, also basic population data for population genetics and anthropological research.     

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.     

Development of a new highly efficient 17 X‐STR multiplex for forensic purposes

Currently, two of the most widely used X‐chromosome STR (X‐STR) multiplexes are composed by ten (GHEP‐ISFG decaplex) and 12 markers (Investigator Argus X‐12 Kit). The number of markers included is a drawback for complex relative testing cases, likewise the large size of some amplicons difficult their application to degraded samples. Here, we present a new multiplex of 17 X‐STRs with the aim of increasing both the resolution power and forensic applicability. This newly proposed set includes the X‐STRs of the GHEP‐ISFG decaplex, four X‐STRs from the Investigator Argus X‐12 Kit, three of them also included in the decaplex, and six additional more. In order to ensure the allele designation, an allelic ladder was developed. The validation of the present multiplex was carried out according to the revised guidelines by the SWGDAM (Scientific Working Group on DNA Analysis Methods). A total of 488 unrelated individuals from four different continents were analyzed. The forensic efficiency evaluation showed high values of combined power of discrimination in males (≥0.999999996) and females (≥0.999999999999995) as well as combined paternity exclusion probabilities in trios (≥0.99999998) and duos (≥0.999996). The results presented herein have demonstrated that the new 17 X‐STR set constitutes a high‐resolution alternative to the current X‐STR multiplexes.     

A targeted ancestry informative InDels panel on capillary electrophoresis for ancestry inference in Asian populations

CE is the primary methodology used in forensic DNA typing. Alleles of commonly used types of genetic markers could be separated and detected via CE based on dye color and migration time. Insertion/deletion (InDel) is an ideal genetic marker for forensic DNA analysis due to their abundance in the human genome, low mutation rate, availability of their allele types via CE, and elimination of stutter peaks. Moreover, InDels could be used as ancestry informative markers since allele frequencies of InDels is different among geographically separated populations. Several ancestry informative insertion/deletion panels have been established based on CE platform to achieve the intercontinental populations distinction. However, improvements to differentiate intracontinental populations is few. In this study, 21 InDels with fixation index (F_ST) > 0.15 were selected and assembled into one ancestry informative insertion/deletion panel. Using well-designed primers, those 21 InDels could be amplified successfully and genotyped on the CE platform accurately and completely. The panel showed a large F_ST distance distinction among the ten Asian populations. Using clustering analysis, ten Asian populations were classified into three subgroups: East Asian, Southeast Asian, and South Asian subgroups. To evaluate the panel's capability in ancestry inference, a validation experiment was undertaken with 319 individuals from four geographically separated populations in China. Four Chinese populations were classified into different ancestry subgroups and 81.8% test individuals’ ancestry could be inferred correctly. Our result showed that development of high ancestry informative InDels panel based on CE platform is a potential for individual ancestry inference among intracontinental populations.     

Sizing short tandem repeat alleles in capillary array gel electrophoresis instruments.

A 96-capillary array gel electrophoresis Applied Biosystems 3700 instrument has been used to analyse AMPF/STR SGM Plus short tandem repeat (STR) loci for forensic applications. This multiplex consists of ten STR loci plus the Amelogenin locus and currently forms the basis of the UK National DNA database that currently holds more than 1 million profiles. Of particular interest is the accuracy of allele designation that is determined by comparison with standard control allelic ladder markers. Some loci have higher standard deviations than others. In particular the high-molecular-weight HUMFIBRA alleles have high standard deviations of the order of 0.15 and it is these alleles that are most likely to be misdesignated. However, this risk is minimised by the analysis of at least five different allelic ladders across the array to estimate the mean size of each allele. In conjunction with this, a series of guidelines that can be programmed into expert systems are used to minimise risks of misdesignation. The efficacy of the procedures utilised are tested by computer simulation and demonstrated to be robust.     

Genetic polymorphisms of 20 short tandem repeat loci from the Han population in Henan,China

The aim of this study was to investigate the genetic polymorphism of 20 short tandem repeat (STR) loci including D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA in Han population of Henan, China and to assess its value in forensic science. Genomic DNA was extracted from 274 blood samples of unrelated healthy individuals in the Henan Han population. Alleles were amplified with PowerPlex® 21 system kit and PCR products were detected with ABI3130 genetic analyzer (Applied Biosystems) and the data were analyzed with modified PowerStats v1.2. A total of 229 alleles were observed in this Han population and the allelic frequencies ranged from 0.0020 to 0.5090 in the present study. Observed genotype distributions for each locus do not show deviations from Hardy–Weinberg equilibrium expectations (p < 0.05). The combined power of discrimination, combined power of exclusion, and combined matching probability of this 20 STR loci were 0.999999999, 0.999999994603, and 4.0433 × 10^?24, respectively. The 20 STR loci are highly polymorphic in the Han population of Henan, China and they may be of great value in forensic science and human population genetics.     

An efficient ancestry informative SNPs panel for further discriminating East Asian populations

In forensic genetics, the use of ancestry informative single-nucleotide polymorphisms (AISNPs) panels can narrow the direction of the investigation by estimating an individual's biogeographic ancestry. However, distinguishing subgroups within continental regions requires more specific panels. In this study, we screened 19 AISNPs from the 1000 Genomes Project (1KG) based on their F_ST values to distinguish target populations in East Asia and obtained genotypes through SNaPshot. The 19 AISNPs could divide the global population of the 1KG into five clusters and could further divide the East Asian population into four clusters: Japanese, Han Chinese, Dai Chinese, and Kinh in Ho Chi Minh City of Vietnam. In summary, the 19-AISNP panel may serve as a useful and cost-effective tool for forensic ancestry inference in East Asian populations at a finer scale.     

A new autosomal STR nineplex for canine identification and parentage testing

A single multiplex PCR assay capable of simultaneously amplifying nine canine‐specific autosomal STR markers (FH3210, FH3241, FH2004, FH2658, FH4012, REN214L11, FH2010, FH2361 and the newly described C38) was developed for individual identification and parentage testing in domestic dogs. In order to increase genotyping efficiency, amplicon sizes were optimized for a 90–350 bp range, with fluorescently labelled primers for use in Applied Biosystems, Inc., platforms. The performance of this new multiplex system was tested in 113 individuals from a case‐study population and 12 random dogs from mixed‐breed origin. Co‐dominant inheritance of STR alleles was investigated in 101 father, mother and son trios. Expected heterozygosity values vary between 0.5648 for REN214L11 and 0.9050 for C38. The high level of genetic diversity observed for most markers provides this multiplex with a very high discriminating power (matching probability=1.63/10¹⁰ and matching probability among siblings=4.9/10³). Allele sequences and a proposal for standardized nomenclature are also herein presented, aiming at implementing the use of this system in forensic DNA typing and population genetic studies. This approach resulted in an optimized and well‐characterized canine DNA genotyping system that is highly performing and straightforward to integrate and employ routinely. Although this STR multiplex was developed for use and tested in a case‐study population, the Portuguese breed Cão de Gado Transmontano, it proved to be useful for general identification purposes or parentage testing.     

Distinguishing three distinct biogeographic regions with an in‐house developed 39‐AIM‐InDel panel and further admixture proportion estimation for Uyghurs

In the forensic field, ancestry‐informative markers (AIMs) showing remarkable allele frequency discrepancies can be useful in deducing the likely ancestral origin of a person or estimating the ancestry component proportions of an admixed population or individual. Diallelic single nucleotide polymorphisms are genetic markers commonly used for ancestry inference, but the genotyping methods of single nucleotide polymorphisms fail to fulfil the demands of cost‐effectiveness and simplicity of experimental manipulation. To overcome the limitations, a 39 ancestry‐informative insertion/deletion polymorphism multiplex panel was developed in the present study to perform ancestry assignment of individuals from three distinct biogeographic regions (Africa, Europe, East Asia). And in the panel design, we also attempted to incorporate AIM‐insertion/deletion polymorphisms exhibiting allelic frequency differences in Han, Uyghur, and Tibetan populations into the multiplex assay, further expecting to provide valuable information for refining ancestry inference within Chinese populations. Statistical analyses were performed to estimate efficiency of this panel in clustering individuals from three continents mentioned above into their corresponding populations, which indicated the potential of the panel in ancestry inference. Besides, we also estimated the ancestral component proportions of Uyghur group and STRUCTURE analysis revealed that Uyghurs from Urumchi city of northern Xinjiang exhibited a distinctly admixed pattern of East Asian and European ancestry components with a ratio of 49:44, reflecting the relatively higher East Asian ancestry component contribution in the gene pool of the Uyghur group.