Development of a novel forensic STR multiplex for ancestry analysis and extended identity testing

There is growing interest in developing additional DNA typing techniques to provide better investigative leads in forensic analysis. These include inference of genetic ancestry and prediction of common physical characteristics of DNA donors. To date, forensic ancestry analysis has centered on population‐divergent SNPs but these binary loci cannot reliably detect DNA mixtures, common in forensic samples. Furthermore, STR genotypes, forming the principal DNA profiling system, are not routinely combined with forensic SNPs to strengthen frequency data available for ancestry inference. We report development of a 12‐STR multiplex composed of ancestry informative marker STRs (AIM‐STRs) selected from 434 tetranucleotide repeat loci. We adapted our online Bayesian classifier for AIM‐SNPs: Snipper, to handle multiallele STR data using frequency‐based training sets. We assessed the ability of the 12‐plex AIM‐STRs to differentiate CEPH Human Genome Diversity Panel populations, plus their informativeness combined with established forensic STRs and AIM‐SNPs. We found combining STRs and SNPs improves the success rate of ancestry assignments while providing a reliable mixture detection system lacking from SNP analysis alone. As the 12 STRs generally show a broad range of alleles in all populations, they provide highly informative supplementary STRs for extended relationship testing and identification of missing persons with incomplete reference pedigrees. Lastly, mixed marker approaches (combining STRs with binary loci) for simple ancestry inference tests beyond forensic analysis bring advantages and we discuss the genotyping options available.     

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.     

Template tailoring: Accurate determination of heterozygous alleles using peptide nucleic acid and dideoxyNTP

Measurement of the length of DNA fragments plays a pivotal role in genetic mapping, disease diagnostics, human identification and forensic applications. PCR followed by electrophoresis is used for DNA length measurement of STRs, a process that requires labeled primers and allelic ladders as standards to avoid machine error. Sequencing‐based approaches can be used for STR analysis to eliminate the requirement of labeled primers and allelic ladder. However, the limiting factor with this approach is unsynchronized polymerization in heterozygous sample analysis, in which alleles with different lengths can lead to imbalanced heterozygote peak height ratios. We have developed a rapid DNA length measurement method using peptide nucleic acid and dideoxy dNTPs to “tailor” DNA templates for accurate sequencing to overcome this hurdle. We also devised an accelerated “dyad” pyrosequencing strategy, such that the combined approach can be used as a faster, more accurate alternative to de novo sequencing. Dyad sequencing interrogates two bases at a time by allowing the polymerase to incorporate two nucleotides to DNA template, cutting the analysis time in half. In addition, for the first time, we show the effect of peptide nucleic acid as a blocking probe to stop polymerization, which is essential to analyze the heterozygous samples by sequencing. This approach provides a new platform for rapid and cost‐effective DNA length measurement for STRs and resequencing of small DNA fragments.     

Short tandem repeat analysis in Japanese population

Short tandem repeats (STRs), known as microsatellites, are one of the most informative genetic markers for characterizing biological materials. Because of the relatively small size of STR alleles (generally 100-350 nucleotides), amplification by polymerase chain reaction (PCR) is relatively easy, affording a high sensitivity of detection. In addition, STR loci can be amplified simultaneously in a multiplex PCR. Thus, substantial information can be obtained in a single analysis with the benefits of using less template DNA, reducing labor, and reducing the contamination. We investigated 14 STR loci in a Japanese population living in Sendai by three multiplex PCR kits, GenePrint PowerPlex 1.1 and 2.2. Fluorescent STR System (Promega, Madison, WI, USA) and AmpF/STR Profiler (Perkin-Elmer, Norwalk, CT, USA). Genomic DNA was extracted using sodium dodecyl sulfate (SDS) proteinase K or Chelex 100 treatment followed by the phenol/chloroform extraction. PCR was performed according to the manufacturer's protocols. Electrophoresis was carried out on an ABI 377 sequencer and the alleles were determined by GeneScan 2.0.2 software (Perkin-Elmer). In 14 STRs loci, statistical parameters indicated a relatively high rate, and no significant deviation from Hardy-Weinberg equilibrium was detected. We apply this STR system to paternity testing and forensic casework, e.g., personal identification in rape cases. This system is an effective tool in the forensic sciences to obtain information on individual identification.     

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.     

17 to 23: A novel complementary mini Y‐STR panel to extend the Y‐STR databases from 17 to 23 markers for forensic purposes

A Y‐STR multiplex system has been developed with the purpose of complementing the widely used 17 Y‐STR haplotyping (AmpFlSTR Y Filer® PCR Amplification kit) routinely employed in forensic and population genetic studies. This new multiplex system includes six additional STR loci (DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643) to reach the 23 Y‐STR of the PowerPlex® Y23 System. In addition, this kit includes the DYS456 and DYS385 loci for traceability purposes. Male samples from 625 individuals from ten worldwide populations were genotyped, including three sample sets from populations previously published with the 17 Y‐STR system to expand their current data. Validation studies demonstrated good performance of the panel set in terms of concordance, sensitivity, and stability in the presence of inhibitors and artificially degraded DNA. The results obtained for haplotype diversity and discrimination capacity with this multiplex system were considerably high, providing further evidences of the suitability of this novel Y‐STR system for forensic purposes. Thus, the use of this multiplex for samples previously genotyped with 17 Y‐STRs will be an efficient and low‐cost alternative to complete the set of 23 Y‐STRs and improve allele databases for population and forensic purposes.     

A SNaPshot assay for genotyping 44 individual identification single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs), which have relatively low mutation rates and can be genotyped after PCR with shorter amplicons compared with short tandem repeats (STRs), are being considered as potentially useful markers in forensic DNA analysis. Those SNPs with high heterozygosity and low Fst (F-statistics) in human populations are described as individual identification SNPs, which perform the same function as STRs used in forensic routine work. In the present study, we developed a multiplex typing method for analyzing 44 selected individual identification SNPs simultaneously by using multiplex PCR reaction in association with fluorescent labeled single base extension (SBE) technique. PCR primers were designed and the lengths of the amplicons ranged from 69 to 125?bp. The population genetics data of 79 unrelated Chinese individuals for the 44 SNP loci were investigated and a series of experiments were performed to validate the characteristic of the SNP multiplex typing assay, such as sensitivity, species specificity and the performance in paternity testing and analysis of highly degraded samples. The results showed that the 44-SNPs multiplex typing assay could be applied in forensic routine work and provide supplementary data when STRs analysis was partial or failed.     

Diversity study of 12 X‐chromosomal STR loci in Hui ethnic from China

X‐chromosomal STRs (X‐STRs) have been used as complements of autosomal STR application in recent years. In this work, we present population genetic data of 12 X‐STRs including DXS101, DXS10159, DXS10162, DXS10164, DXS6789, DXS7133, DXS7423, DXS7424, DXS8378, DXS981, GATA165B12, and GATA31E08 loci in a sample of 231 unrelated healthy individuals from the Hui ethnic group in Ningxia Hui Autonomous Region, China. Allelic frequencies of the 12 X‐STR loci and haplotypic frequencies of the reported linkage groups (DXS7424‐DXS101 and DXS10159‐DXS10164‐DXS10162) were investigated in the group, respectively. No STR loci showed significant deviations from the Hardy–Weinberg equilibriums and no linkage disequilibriums of pairwise loci were found after Bonferroni correction, respectively. A combined power of discrimination in female individuals was 0.999999999985 and that in male individuals was 0.99999967, respectively. The combined mean exclusion chance in deficiency cases, normal trios and duo cases were 0.999934, 0.995754, and 0.999796, respectively. Significant differences were observed from 0 to 8 loci, when making comparisons between the data of Hui ethnic group and previously reported data from other 16 populations. The results indicated the new panel of 12 X‐STR loci might be useful for forensic science application.     

Structural polymorphism analysis of Chinese Mongolian ethnic group revealed by a new STR panel: Genetic relationship to other groups

Mongolian is the eighth largest ethnic minority on Chinese population data according to the 2010 census. In the present study, we presented the first report about the allelic frequencies and forensic statistical parameters at the 21 new STRs and analyzed linkage disequilibrium of pairwise loci in the Mongolian ethnic minority, China. Hardy–Weinberg equilibrium tests demonstrated no significant deviations except for the D1S1627 locus. The cumulative power of discrimination and power of exclusion of all the loci are 0.9999999999999999992576 and 0.9999997528, respectively. The results of analysis of molecular variance showed that significant differences between the Mongolian and the other eight populations were found at 1‐9 STR loci. In population genetics, the results of principal component analysis, structure analysis, and phylogenetic reconstruction analysis indicated shorter genetic distance between the Mongolian group and the Ningxia Han. All the results suggest that the 21 new STR loci will contribute to Chinese population genetics and forensic caseworks in the Mongolian group.     

Genetic diversity and haplotype structure of 21 Y‐STRs,including nine noncore loci,in South Tunisian Population: Forensic relevance

Y chromosome STRs (Y‐STRs) are being used frequently in forensic laboratories. Previous studies of Y‐STR polymorphisms in different groups of the Tunisian population identified low levels of diversity and discrimination capacity (DC) using various commercial marker sets. This definitely limits the use of such systems for Y‐STRs genotyping in Tunisia. In our investigation on South Tunisia, 200 unrelated males were typed for the 12 conventional Y‐STRs included in the PowerPlex® Y System. Additional set of nine noncore Y‐STRs including DYS446, DYS456, DYS458, DYS388, DYS444, DYS445, DYS449, DYS710, and DYS464 markers were genotyped and evaluated for their potential in improving DC. Allele frequency, gene diversity, haplotype diversity (HD), and DC calculation revealed that DYS464 was the most diverse marker followed by DYS710 and DYS449 markers. The standard panel of 12 Y‐STRs (DC = 80.5%) and the nine markers were combined to obtain DC of 99%. Among the 198 different haplotypes observed, 196 haplotypes were unique (HD = 99.999). Out of the nine noncore set, six Y‐STRs (DYS458, DYS456, DYS449, DYS710, DYS444, and DYS464) had the greatest impact on enhancing DC. Our data provided putative Y‐STRs combination to be used for genetic and forensic applications.     

Evaluation of the protective capabilities of nucleosome STRs obtained by large‐scale sequencing

Partial DNA profiles are often obtained from degraded forensic samples and are hard to analyze and interpret. With in‐depth studies on degraded DNA, an increasing number of forensic scientists have focused on the intrinsic structural properties of DNA. In theory, nucleosomes offer protection to the bound DNA by limiting access to enzymes. In our study, we performed large‐scale DNA sequencing on nucleosome core DNA of human leucocytes. Five nucleosome short tandem repeats (STRs) were selected (including three forensic common STRs (i.e. TPOX, TH01, and D10S1248) and two unpublished STRs (i.e. AC012568.7 and AC007160.3)). We performed a population genetic investigation and forensic genetic statistical analysis of these two unpublished loci on 108 healthy unrelated individuals of the HeBei Han population in China. We estimated the protective capabilities of five selected nucleosome loci and MiniFiler? loci with artificial degraded DNA and case samples. We also analyzed differences between sequencing results and software predicted results. Our findings showed that nucleosome STRs were more likely to be detected than MiniFiler? loci. They were well protected from degradation by nucleosomes and could be candidates for further nucleosome multiplex construction, which would increase the chances of obtaining a better balanced profile with fewer allelic drop‐outs.     

Analysis of 17 Y‐STR loci haplotype and Y‐chromosome haplogroup distribution in five Chinese ethnic groups

To investigate genetic diversity in Chinese populations, 706 unrelated male individuals from five ethnic groups (Han, Korean, Hui, Mongolian, and Tibetan, respectively) were analyzed with 17 Y‐chromosomal STRs. The haplotype diversity was 0.99985 in the combined data. A total of 675 distinct haplotypes were observed, of which 649 were unique. Y‐chromosome haplogroups in the five groups were also predicted with Y‐STR haplotypes. Genetic distance between the five studied ethnic groups and other published groups was analyzed by analysis of molecular variance and visualized in a multidimensional scaling plot. In conclusion, the 17 Y‐STR loci are highly polymorphic markers in the five groups and hence are very useful in forensic application, population genetics, and human evolution studies.     

Genetic diversity and haplotype structure of 24 Y‐chromosomal STR in Chinese Hui ethnic group and its genetic relationships with other populations

In the present study, 24 Y‐chromosomal short tandem repeat (Y‐STR) loci were analyzed in 115 unrelated Hui male individuals from Haiyuan county or Tongxin county, Ningxia Hui Autonomous Region, China, to evaluate the forensic application of the 24 STR loci and to analyze interpopulation differentiations by making comparisons between the Hui group data and previously published data of other 13 populations. A total of 115 different haplotypes were observed on these 24 Y‐STR loci. The gene diversities ranged from 0.4049 (DYS437) to 0.9729 (DYS385a, b). The overall haplotype diversity was 1 at AGCU 24 Y‐STR loci level, while the values were reduced to 0.999237, 0.996949, and 0.996644 at the Y‐filer 17 loci, 11 Y‐STR loci of extended haplotype and 9 Y‐STR loci of minimal haplotype levels, respectively; whereas, haplotype diversity for additional 7 loci (not included in Y‐filer 17 loci) was 0.995271. The pairwise F_ST, multidimensional scaling plot and neighbor‐joining tree indicated the Hui group had the closest genetic relationship with Sala in the paternal lineage in the present study. In summary, the results in our study indicated the 24 Y‐STRs had a high level of polymorphism in Hui group and hence could be a powerful tool for forensic application and population genetic study.     

Evaluation and validation of the ABI 3700, ABI 3100, and the MegaBACE 1000 capillary array electrophoresis instruments for use with short tandem repeat microsatellite typing in a forensic environment

The demand for high-throughput DNA profiling has increased with the introduction of national DNA databases and has led to the development of automated methods of short tandem repeat (STR) profile production; however, a potential bottleneck still exists at the gel electrophoresis stage. Capillary electrophoresis sequencers capable of processing 96 samples with considerably reduced manual intervention are now available. In this paper, we compare the ABI Prism 377 slab-gel sequencer currently used by the Forensic Science Service with three leading capillary array electrophoresis instruments: the ABI Prism 3700, the Amersham MegaBACE 1000 and the 16-capillary ABI Prism 3100. We describe the experiments used to evaluate and validate these platforms for forensic use with the STR multiplex Ampf/STR SGMplus [1, 2], along with comparative data from the ABI Prism 377 sequencer.     

Genetic profile characterization and population study of 21 autosomal STR in Chinese Kazak ethnic minority group

Short tandem repeat loci have been recognized as useful tools in the routine forensic application and in recent decades, more and more new short tandem repeat (STR) loci have been constantly discovered, studied, and applied in forensic caseworks. In this study, we investigated the genetic polymorphisms of 21 STR loci in the Kazak ethnic minority as well as the genetic relationships between the Kazak ethnic minority and other populations. Allelic frequencies of 21 STR loci were obtained from 114 unrelated healthy Kazak individuals in the Ili Kazak Autonomous Prefecture, Xinjiang Uigur Autonomous Region of China. We observed a total of 159 alleles in the group with the allelic diversity values ranging from 0.0044 to 0.5088. The highest polymorphism was found at D19S433 locus and the lowest was found at D1S1627. Statistical analysis of the generated data indicated no deviation from Hardy–Weinberg equilibriums at all 21 STR loci. In order to estimate the population differentiation, allelic frequencies of all STR loci of the Kazak were compared with those of other neighboring populations using analysis of molecular variance method. Statistically significant differences were found between the studied population and other populations at 2–7 STR loci. A neighbor‐joining tree was constructed based on allelic frequencies of the 21 STR loci and phylogenetic analysis indicates that the Kazak has a close genetic relationship with the Uigur ethnic group. The present results may provide useful information for forensic sciences and population genetics studies, and can also increase our understanding of the genetic background of this group. The present findings showed that all the 21 STR loci are highly genetically polymorphic in the Kazak group, which provided valuable population genetic data for the genetic information study, forensic human individual identification, and paternity tests.     

A powerful,novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations

A new multiplex system for six tetranucleotide short tandem repeat (STR) loci was devised based on multicolor dye technology. Six loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), each with high discriminating power (each unbiased estimates of expected heterozygosity, Exp. Hz, > 0.80 in a preliminary test), were selected from more than 100 tetranucleotide STRs included in a commercially available primer set. These loci were also selected so as not to link with general STRs in commercially released kits including the combined DNA index system (CODIS) 13 core STRs. The primers were newly designed in the present study, in which each amplicon size had a range of less than 200 base pairs (bp), in order to genotype from highly degraded template DNA. Using this system, we genotyped 270 Honshu (mainland)-Japanese and 187 Okinawa-Japanese. From these allele frequencies, we performed three tests, a homozygosity test, a likelihood ratio test and an exact test for Hardy-Weinberg equilibrium (HWE), and no significant deviations (p < 0.05) from HWE were observed. We also compared the allele distributions at six STRs between both populations, and they were significantly different (p < 0.05) at three loci (D6S2439, D9S1118 and D4S2639). Furthermore, the Exp. Hz and the power of discrimination (PD) at all loci in the Honshu-Japanese population were higher than 0.80 and 0.93, respectively. These statistical values for discriminating power in the Honshu-Japanese were almost the same as in the Okinawa-Japanese. This novel, multiplex polymerase chain reaction (PCR) amplification and typing system for six STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field.     

Unknown biological mixtures evaluation using STR analytical quantification

Allelic quantification of STRs, where the presence of three or more alleles represents mixtures, provides a novel method to identify mixtures from unknown biological sources. The allelic stutters resulting in slightly different repeat containing products during fragment amplification can be mistaken for true alleles complicating a simple approach to mixture analysis. An algorithm based on the array of estimated stutters from known samples was developed and tuned to maximize the identification of true non-mixtures through the analysis of three pentanucleotide STRs. Laboratory simulated scenarios of needle sharing generated 58 mixture and 38 non-mixture samples that were blinded for determining the number of alleles. Through developing and applying an algorithm that additively estimates stuttering around the two highest peaks, mixtures and non-mixtures were characterized with sensitivity of 77.5, 82.7 and 58% while maintaining the high specificity of 100, 97.4 and 100 for the W, X, and Z STRs individually. When all three STRs were used collectively, the resulting sensitivity and specificity was 91.4 and 97.4%, respectively. The newly validated approach of using multiple STRs as highly informative biomarkers in unknown sample mixture analyses has potential applications in genetics, forensic science, and epidemiological studies.     

Development and validation of a new 18 X-STR typing assay for forensic applications

With a unique inheritance pattern compared to autosomal short tandem repeats (A-STRs), X chromosomal STRs (X-STRs) have special usage in forensic relationship testing. In this study, we designed a multiplex amplification system (named TYPER-X19 multiplex assay) consisting of 18 STR loci spreading from 7.837 to 149.460 Mb on the X chromosomes (DXS9895, DXS8378, DXS9902, DXS6810, DXS7132, DXS10079, DXS6789, DXS7424, DXS101, DXS6797, DXS7133, DXS6804, GATA165B12, DXS10103, HPRTB, GATA31E08, DXS8377, and DXS7423), and the amelogenin. PCR primers were marked with four kinds of fluorophores including FAM, HEX, TAMRA, and ROX. The multiplex system was optimized and tested for precision, concordance, reproducibility, sensitivity, stability, DNA mixture, and species specificity according to the conventional validation guidelines. The results indicated that the system was accurate, reliable, and sensitive enough, and was suitable for common forensic case-type samples. In the population genetic study, a total of 148 alleles were detected at the 18 X-STR loci in 398 Southern Han Chinese. Relatively high combined power of discrimination in male (PD_m), power of discrimination in female (PD_f), mean paternity exclusion chance in trios (MEC_trio), and mean paternity exclusion chance in duos (MEC_Duo) by Desmarais were detected, and HPRTB-DXS10103 was in linkage disequilibrium. The results suggested that the TYPER-X19 multiplex assay was suitable for forensic applications.     

Recombination analysis of autosomal short tandem repeats in Chinese Han families

Recombination fractions between forensic STRs can be extrapolated from the International HapMap Project, but the concordance between recombination fractions predicated from genetic maps and derived from observation of STR transmissions in families is still ambiguous for autosomal STRs because of limited family studies. Therefore, the main goal of this study is to compare recombination fractions estimated by pedigree analysis with those derived from HapMap phase SNP data. Genotypes of nine autosomal STR pairs (TPOX‐D2S1772, D5S818‐CSF1PO, D7S3048‐D7S820, D8S1132‐D8S1179, TH01‐D11S2368, vWA‐D12S391, D13S325‐D13S317, D18S51‐D18S1364, and D21S11‐PentaD) from 207 two‐generation families with two to five children (the number of families with five, four, three, and two children was 2, 3, 20, and 182, respectively) were used to analyze the recombination. The linkage analysis showed that significant linkage was observed at six STR pairs (D5S818‐CSF1PO, D8S1132‐D8S1179, TH01‐D11S2368, vWA‐D12S391, D13S325‐D13S317, and D18S51‐D18S1364) with genetic distances <36.22 cM in HapMap. Their recombination fractions calculated from family data were very close to those derived from HapMap. However, three STR pairs of TPOX‐D2S1772, D7S3048‐D7S820, and D21S11‐PentaD showed no significant linkage with genetic distances from 43.38 to 91.49 cM. Our results indicate that recombination fractions extrapolated from HapMap can provide a substitute if empirical data are unavailable for the linkage STR pair with a genetic distance spanned <36.22 cM.     

Expansion of a SNaPshot assay to a 55‐SNP multiplex: Assay enhancements,validation, and power in forensic science

A previously developed multiplex assay with 44 individual identification SNPs was expanded to a 55plex assay. Fifty‐four highly informative SNPs and an amelogenin sex marker were amplified in one PCR reaction and then detected with two SNaPshot reactions using CE. PCR primers for four loci, 28 single‐base extension primers, and the reaction conditions were altered to improve the robustness of the method. A detailed approach for allele calling was developed to guide analysis of the electropherogram. One hundred and eighty unrelated individuals and 100 father‐child‐mother trios of the Han population in Hebei, China were analyzed. No mutation was found in the SNP loci. The combined mean match probability and cumulative probability of exclusion were 1.327 × 10^?22 and 0.999932, respectively. Analysis of the 54 SNPs and 26 STRs (included in the AmpFLSTR Identifiler and Investigator HDplex kits) showed no significant linkage disequilibriums. Our research shows that the expanded SNP multiplex assay is an easily performed and valuable method to supplement STR analysis.