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.     

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.     

Exploring of tri-allelic SNPs using pyrosequencing and the SNaPshot methods for forensic application

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.     

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.     

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.     

A new multiplex for human identification using insertion/deletion polymorphisms

Human identification is usually based on the study of STRs or SNPs depending on the particular characteristics of the investigation. However, other types of genetic variation such as insertion/deletion polymorphisms (indels) have considerable potential in the field of identification, since they can combine the desirable characteristics of both STRs and SNPs. In this study, a set of 38 non‐coding bi‐allelic autosomal indels reported to be polymorphic in African, European, and Asian populations were selected. We developed a sensitive genotyping assay, which is able to characterize all 38 bi‐allelic markers using a single multiplex PCR and detected with standard CE analyzers. Amplicon length was designed to be shorter than 160 bp. Complete profiles were obtained using 0.3 ng of DNA, and full genotyping of degraded samples was possible in cases where standard STR typing had partially failed. A total of 306 individuals from Angola, Mozambique, Portugal, Macau, and Taiwan were studied and population data are presented. All indels were polymorphic in the three population groups studied and the random match probabilities of the set ranged in orders of magnitude from 10^?14 to 10^?15. Therefore, the indel‐plex represents a valuable approach in human identification studies, especially in challenging DNA cases, as a more straightforward and efficient alternative to SNP typing.     

Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies

We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.     

连接反应介导的等位基因特异性扩增-微流控芯片电泳法同时检测多个SNP位点

以CYP2D6基因中的6个SNP位点为测定对象, 开展多个SNP位点同时测定的方法学研究.     

Improved adapter-ligation-mediated allele-specific amplification for multiplex genotyping by using software

Adapter-ligation-mediated allele-specific amplification (ALM-ASA) is a potential method for multiplex SNPs typing at an ultra low cost. Here, we describe a kind of software, which designs allele-specific primers for ALM-ASA assay on multiplex SNPs. DNA sequences containing SNPs of interest are submitted into the software which contains various endonucleases for options. Based on the SNP sequence information and the selected endonucleases, the software is capable of automatically generating sets of information needed to perform genotyping experiments. Each set contains a suitable endonuclease, qualified allele-specific primers with orientations and melting temperatures, sizes of allele-specific amplicons, and gel electropherograms simulated according to the sizes of the allele-specific amplicons and the mobility of DNA fragments in 2% agarose gel. Seven SNPs in the arylamines N-acetyltransferase 2 (NAT2) gene, five SNPs in the BRCA1 gene, five SNPs in the COMT gene, six SNPs in the CYP2E1 gene, five SNPs in the MPO gene, and six SNPs in the NRG1 gene were selected for evaluating the software. Without extra optimization, seven SNPs in the NAT2 gene were successfully genotyped for genomic DNA samples from 127 individuals by using the first set of allele-specific primers yielded by the software. Although several steps are used in the ALM-ASA assay, the whole genotyping process can be completed within 3 h by optimizing each step. Profiting from the software, the ALM-ASA assay is easy-to-perform, labor-saving, and accurate.     

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.     

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 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.     

基于微流控芯片的72重单核苷酸多态性族群推断系统的构建

建立了常染色体单核苷酸多态性（SNPs）复合检测芯片体系,用于未知个体的族群来源推断。基于前期筛选的74-SNPs组合,采用竞争性等位基因特异性聚合酶链式反应（PCR）的原理构建SNPs的扩增体系,在微流控芯片的每个反应孔内完成一个SNP的检测,通过高通量PCR微流控芯片实现了其中72个SNPs的同步检测。芯片的扩增由平板PCR仪完成,反应孔的荧光信号通过激光共聚焦扫描仪检测,最终通过提取的荧光值进行结果分析。使用该芯片检测获得52份样本的SNPs分型,分型结果的准确率为100%。以57个人群的3628个样本为参考人群数据库,进行20份样本的族群来源推断,推断结果与样本的实际来源一致。本研究建立的常染色体72个SNPs微流控芯片体系可以有效地进行SNP多态性分析检测,基于参考数据库,20份检测样本族群推断的准确性为100%。     

Analysis of four PCR/SNaPshot multiplex assays analyzing 52 SNPforID markers

The SNPfor ID consortium identified a panel of 52 SNPs for forensic analysis that has been used by several laboratories worldwide. The original analysis of the 52 SNPs was based on a single multiplex reaction followed by two single‐base‐extension (SBE) reactions each of which was analyzed using capillary electrophoresis. The SBE assays were designed for high throughput genetic analyzers and were difficult to use on the single capillary ABI PRISM 310 Genetic Analyzer and the latest generation 3500 Genetic Analyzer, as sensitivity on the 310 was low and separation of products on the 3500 with POP‐7™ was poor. We have modified the original assay and split it into four multiplex reactions, each followed by an SBE assay. These multiplex assays were analyzed using polymer POP‐4™ on ABI 310 PRISM® and polymers POP‐4™, POP‐6™ and POP‐7™ on the 3500 Genetic Analyzer. The assays were sensitive and reproducible with input DNA as low as 60 pg using both the ABI 310 and 3500. In addition, we found that POP‐6™ was most effective with the 3500, based on the parameters that we assessed, achieving better separation of the small SBE products; this conflicted with the recommended use of POP‐7™ by the instrument manufacturer. To support the use of the SNP panel in casework in Malaysia we have created an allele frequency database from 325 individuals, representing the major population groups within Malaysia. Population and forensic parameters were estimated for all populations and its efficacy evaluated using 51 forensic samples from challenging casework.     

Improving the analysis of Y‐SNP haplogroups by a single highly informative 16 SNP multiplex PCR‐minisequencing assay

The SNP haplogroups of the Y‐chromosome are nonrandomly distributed among human populations. They are used for tracing the phylogeographical history of paternal lineages of male individuals and can be a useful tool for approaching the patrilineal bio‐geographic ancestry of unknown forensic evidences. With the aim of facilitating the inference of the principal informative worldwide Y‐SNP haplogroups, we have selected the minimum possible number of key Y‐SNPs to be amplified in a sensitive single multiplex PCR and detected by minisequencing. This assay, that includes 16 Y‐SNPs, was tested for male human specificity, sensitivity, and reproducibility. Its effectiveness was assessed in a set of degraded DNA samples and in a panel of male individuals from different worldwide populations. All these tests demonstrated the convenience of this assay for assigning the major Y haplogroups to forensic evidences by one single PCR‐minisequencing reaction.     

A multiplex SNP genotyping by allele‐specificspecific PCR based on stem‐loop and universal fluorescent primers of Chr1daxin mice

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele‐specific PCR amplification of genomic DNA with two stem‐loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome‐wide association study, pharmacogenetics, and medical diagnostics.     

Analysis of single nucleotide polymorphisms by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for typing single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described, in which a mass-tagged dideoxynucleoside triphosphate is employed in a primer extension reaction in place of an unmodified dideoxynucleoside triphosphate (ddNTP). The increased mass difference due to the presence of the mass-tag greatly facilitates the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous samples. Twenty commercially available mass-tagged dideoxynucleoside triphosphates were screened for amenability to incorporation by AmpliTaq FS and ThermoSequenase DNA polymerases in single nucleotide primer extension (SNuPE) reactions. Several sample preparation and purification methods were also examined and compared. Float dialysis was found to be a simple, versatile, and effective method for purification of the extension products. High specificity and sensitivity were obtained, and all six possible biallelic SNP heterozygotes were determined accurately using a 44-mer synthetic oligonucleotide target DNA as a model system. Further validation of the method was demonstrated in the analysis of five single-base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182-bp PCR amplicons amplified from three plasmid and three human genomic DNA samples were genotyped at five variable positions, with results in 100% concordance with conventional sequencing. Genotypes were determined accurately at five sequence-tagged sites (STSs).     

Dissection of mitochondrial superhaplogroup H using coding region SNPs

Haplogroup H (hg H) includes about 40-50% of the West Eurasian mitochondrial DNA (mtDNA) samples investigated so far. In order to enhance discrimination within this haplogroup we selected 45 coding region SNPs that allow to ascribe samples to the main phylogenetic branches of super hg HV (that embraces hg H) and, in particular, to H sublineages with a much finer resolution than previous studies. SNP selection was carried out using the most up-to-date available literature on population and forensic genetics and extended by means of phylogenetic analysis of complete or coding region genomes (<430) and control region sequences. A meticulous inspection of the H phylogeny led us to the observation of various but uncharacterized subclades of hg H. The selected SNPs were amplified in two PCR-multiplex reactions and subsequently targeted in three single-base extension multiplex reactions. A total of 2214 West Eurasian samples were screened for hg H specific loci 2706 and 7028, of which 859 fell in hg H and were further subjected to subhaplogroup typing. We observed 35 different subhaplogroups in total, 33 of which were found at frequencies below 5%. This assay can be used as a prescreening tool in forensic casework for rapid discrimination between divergent lineages (very effective for high-volume crime cases) or as discriminatory assay, when identical hg H haplotypes were obtained by control region sequencing.     

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.     

A SNaPshot assay for detection of 45 mutations in the SCN5A gene in the Chinese Han Population

Sudden cardiac death (SCD) occurs frequently in forensic practice and results in no visible pathological changes that can be detected in an autopsy. In recent years, the genetic background has been emphasized when examining SCD cases. The aim of this study is to establish a feasible system to detect SCD‐related genes for forensic DNA laboratories. Forty‐five reported SCD‐associated SNPs from sodium voltage‐gated channel alpha subunit 5 (SCN5A) were considered in our experiment. We established a SNaPshot assay for the typing of 45 SNPs using multiplex PCR and the minisequencing technique. Two multiplex PCRs were performed and optimized to cover 14 and 16 DNA fragments. The SCD victims came from the Chinese Han population residing in Shanxi and Chongqing provinces and were examined and compared with a non‐SCD group and with normal healthy individuals. A missense mutation at rs1805124 (H558R) was detected in the Chinese Han population in this study. A SNaPshot assay can be performed in any forensic DNA laboratory and would be capable of meeting the increasing demand for SCD detection. This method would also be beneficial for screening at‐risk in family members of SCD victims.