SNP Miniplexes for Individual Identification of Random‐Bred Domestic Cats

Phenotypic and genotypic characteristics of the cat can be obtained from single nucleotide polymorphisms (SNPs) analyses of fur. This study developed miniplexes using SNPs with high discriminating power for random‐bred domestic cats, focusing on individual and phenotypic identification. Seventy‐eight SNPs were investigated using a multiplex PCR followed by a fluorescently labeled single base extension (SBE) technique (SNaPshot^®). The SNP miniplexes were evaluated for reliability, reproducibility, sensitivity, species specificity, detection limitations, and assignment accuracy. Six SNPplexes were developed containing 39 intergenic SNPs and 26 phenotypic SNPs, including a sex identification marker, ZFXY. The combined random match probability (cRMP) was 6.58 × 10^?19 across all Western cat populations and the likelihood ratio was 1.52 × 10¹⁸. These SNPplexes can distinguish individual cats and their phenotypic traits, which could provide insight into crime reconstructions. A SNP database of 237 cats from 13 worldwide populations is now available for forensic applications.     

Forensic Identification Using a Multiplex Assay of 47 SNPs*

Abstract: As a powerful alternative to short tandem repeat (STR) profiling, we have developed a novel panel of 47 single nucleotide polymorphisms (SNPs) for DNA profiling and ABO genotyping. We selected 42 of the 47 SNPs from a panel of 86 markers that were previously validated as universal individual identification markers and identified five additional SNPs including one gender marker and four ABO loci. Match probability of the 42 validated SNPs was found to be 9.5 × 10^?18 in Han Chinese. SNP analysis correctly assessed a panel of historical cases, including both paternity identifications in trios and individual identifications. In addition, while STR profiling of degraded DNA provided information for 11 loci of 16 potential markers with low peak intensities, SNPstream^® genotyping was sufficient to identify all 47 SNPs. In summary, SNP analysis is equally effective as STR profiling, but appears more suited for individual identification than STR profiling in cases where DNA may be degraded.     

A 21‐Locus Autosomal SNP Multiplex and its Application in Forensic Science

To develop a cost‐effective technique for single‐nucleotide polymorphism (SNP) genotyping and improve the efficiency to analyze degraded DNA, we have established a novel multiplex system including 21‐locus autosomal SNPs and amelogenin locus, which was based on allele‐specific amplification (ASA) and universal reporter primers (URP). The target amplicons for each of the 21 SNPs arranged from 63 base pair (bp) to 192 bp. The system was tested in 539 samples from three ethnic groups (Han, Mongolian, and Zhuang population) in China, and the total power of discrimination (TPD) and cumulative probability of exclusion (CPE) were more than 0.99999999 and 0.98, respectively. The system was further validated with forensic samples and full profiles could be achieved from degraded DNA and 63 case‐type samples. In summary, the multiplex system offers an effective technique for individual identification of forensic samples and is much more efficient in the analysis of degraded DNA compared with standard STR typing.     

Effectiveness of Single‐Nucleotide Polymorphisms to Investigate Cattle Rustling

Short tandem repeats (STR)s have been the eligible markers for forensic animal genetics, despite single‐nucleotide polymorphisms (SNP)s became acceptable. The technology, the type, and amount of markers could limit the investigation in degraded forensic samples. The performance of a 32‐SNP panel genotyped through OpenArrays^TM (real‐time PCR based) was evaluated to resolve cattle‐specific forensic cases. DNA from different biological sources was used, including samples from an alleged instance of cattle rustling. SNPs and STRs performance and repeatability were compared. SNP call rate was variable among sample type (average = 80.18%), while forensic samples showed the lowest value (70.94%). The repeatability obtained (98.7%) supports the used technology. SNPs had better call rates than STRs in 12 of 20 casework samples, while forensic index values were similar for both panels. In conclusion, the 32‐SNPs used are as informative as the standard bovine STR battery and hence are suitable to resolve cattle rustling investigations.     

16个线粒体DNASNP复合检测体系的建立

目的建立一种复合检测线粒体DNA（mtDNA）单核苷酸多态性（SNP）分型的方法。方法通过设计等位基因特异性引物并结合毛细管电泳分型技术平台,建立包含16个mtDNASNP位点的复合扩增检测体系。对50名汉族无关个体血样进行mtDNASNP检测,并通过直接测序法对其分型结果进行验证。结果50个样本经本检测体系复合扩增后,均得到清晰的SNP分型结果,当模板量在0．5～10pg时能得到较理想的分型图谱。样本的复合检测结果与直接测序法结果完全一致。结论建立的复合检测体系检测mtDNASNP方法灵敏度高、操作简便、分型准确,为针对mtDNA进行简便、有效的中高通量多态性分型提供了一种新方法。     

Typing of 49 autosomal SNPs by SNaPshot in the Slovenian population

A total of 157 unrelated individuals residing in Slovenia were typed for 49 of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex with the SNaPshot^® assay. We obtained full SNP profiles in all but one individual and perfect concordance was obtained in duplicated analyses. Allele frequencies are presented for the 49 SNPs. No deviation from HWE was observed for any SNP. F_IS and F_ST were estimated. A principal coordinate analysis performed on six populations (Slovenian, Danish, Somali, Greenland, Turkish and Chinese) showed that the Slovenian population grouped with the Danish population. The mean power of discrimination for the Slovenian population was 1.1 × 10⁻¹⁹, and the mean exclusion probability for trios was 99.96%.     

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot™ assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.     

Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot EZ1 Workstation

FTA Cards (GE Healthcare) have been used for more than 4 years in Denmark for the collection of buccal cells as reference samples in crime cases. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used. In average, full STR profiles were generated from approximately 95% of the FTA Cards with a standard punching protocol, while partial or no STR profile were obtained from 5% of the samples. Here, the Qiagen BioRobot^® EZ1 Workstation (Qiagen) and the EZ1 DNA Investigator Kit (Qiagen) was used to extract DNA from 29 FTA Cards from which a complete STR profile was not generated with the standard punching protocol. All 29 samples were successfully typed with the AmpF?STR^® Identifiler™ PCR Amplification Kit (Applied Biosystems) and with the SNPforID 49plex SNP assay. The lowest amount of DNA that resulted in complete STR and SNP profiles was 80 pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.     

Melting curve SNP (McSNP) genotyping: a useful approach for diallelic genotyping in forensic science

The increasing availability of Single Nucleotide Polymorphisms (SNPs) and Deletion/Insertion Polymorphisms (DIPs), as well as the outstanding progress in SNP genotyping technologies, will impact forensics profoundly. We have developed a new method for genotyping SNPs and DIPs, which is based on the determination of melting curve profiles of amplified DNA in solution. We have termed this method Melting curve SNP (McSNP) genotyping. Melting curve profiles are composites of the particular melting temperatures (Tm) of the individual fragments that comprise the DNA sample. Simple mixtures of DNA can be resolved in a very robust and efficient fashion, since the samples can be scored in the plates in which they were amplified with no or very few post-PCR manipulations. As such, McSNP is one of the least expensive genotyping methods available and can and should be useful in forensic science.     

福尔马林固定组织SNP与STR检测的比较

目的检测经长期福尔马林固定的组织降解情况,并比较组织中SNP与STR的检出率。方法本文对24例经福尔马林固定、-20℃保存5年的组织样本,采用Quantifiler?Trio DNA定量试剂盒检测样本DNA的降解系数及浓度,运用55-SNPs SNa Pshot复合分型体系和Power Plex?21试剂盒分别进行SNP与STR检测。结果大部分样本降解系数在1~8,发生不同程度的降解。与未降解样本相比,SNP分型完全一致,检出率为100%;其中8例样本STR分型存在33个等位基因丢失,降解系数均大于2.6,且75.8%的等位基因片段长度大于300bp。当样本检测出16个STR基因座时,似然率与54个SNP相当。当样本检出大于17个STR时,似然率大于54个SNP。STR基因座片段长度与等位基因检出率之间呈负相关。除2例样本降解系数较小却发生等位基因丢失外,其余样本降解系数与等位基因检出率之间呈负相关。结论经福尔马林长期固定的组织DNA易降解,检测SNP明显优于STR,但需要更多的SNP以提高个体识别能力。     

Genome‐wide Screen for Individual Identification SNPs (IISNPs) and the Confirmation of Six of Them in Han Chinese with Pyrosequencing Technology*

Abstract: Single nucleotide polymorphisms (SNPs) offer promise to forensic DNA analysts, but it remains uncertain whether a panel of individual identification SNPs can be as informative as the Combined DNA Index System short tandem repeats. Based on the highly accurate and publicly available HapMap SNP database (r21a) and a minor allele frequency cutoff of ≥0.45, we completed a genome‐wide screen through 3,905,819 SNPs with internally modified computer programs and identified 1439 SNPs with high heterozygosity and low F_st values among four populations (Utah Caucasian, Han Chinese, Tokyo Japanese, and Nigerian Yoruba). Using pyrosequencing technology, we studied six loci in a relatively large group of samples to determine whether these loci were as informative as the HapMap data suggest. These SNPs performed as expected in the Han Chinese in terms of heterozygosity and F_st. The 1439 identified SNPs should provide a comprehensive and reliable set of loci for identity and relationship testing.     

Internal validation of 29 autosomal SNP multiplex using a ABI 310 genetic analyser

In the last few years genetic identification and paternity testing have begun to make increasing use of autosomal SNP (Single Nucleotide Polymorphism) typing as a supplement or alternative to STR analysis. With the improvement in detection technology SNP analysis is likely to be easier and more sensitive, with the generation of new methods and multiplex systems for a growing array of SNP markers. SNPforID consortium developed 52 SNP PCR multiplex for human identification purposes detected with 23 plex and 29 plex single base extension reactions (Auto1 and 2 respectively). In this study, internal validation for the 29 SNPs of Auto2 was carried out by performing a 29 plex PCR and single base extension reaction on control samples and previously analyzed forensic casework and subsequent detection with an AB 310 Genetic Analyzer. We tested the accuracy, precision, sensitivity and reproducibility of the Auto2 multiplex with this instrument in our laboratory. We used 9947A control DNA samples of the AmpF?STR Identifiler™ kit to test the validation parameters together with non-probative DNA samples from whole blood and buccal swab samples of 29 healthy donors from different parts of Istanbul. Good results were obtained but interpretation of the peak patterns obtained on the AB 310 requires care and thorough optimization before they can be readily compard to those obtained from multiple capillary AB 31xx Analyzers. We succesfully optimized and validated the SNPforID Auto2 multiplex system for identification analyses in our laboratory.     

Uses of the NIST 26plex STR assay for human identity testing

Ongoing work at the U.S. National Institute of Standards and Technology has focused on the characterization of 26 autosomal STR loci for human identity testing. These 26 loci are in addition to the existing 13 U.S. core loci and those found in PowerPlex16 and Identifiler commercial STR typing kits. The amplification of the 26 loci has been optimized for degraded extracts in unique miniplex panels and also for reference samples as a single reaction 26plex assay. A study has been performed comparing genotypes obtained with the 26plex primers to those with miniplex panels for allele drop out and concordance. The forensic utility of the 26plex assay was evaluated for situations where additional loci are beneficial. The utility of this large multiplex was also tested in a case involving DNA extracted from degraded bone samples. The 26plex can serve as a low-cost assay (compared to commercially available kits) useful for both sorting comingled remains and providing additional markers for increased statistical support for samples that require “non-trio” family references for human identification.     

Using FastID to analyze complex SNP mixtures from indoor dust

Forensically relevant single nucleotide polymorphisms (SNPs) can provide valuable supplemental information to short tandem repeats (STRs) for investigative leads, and genotyping can now be streamlined using massively parallel sequencing (MPS). Dust is an attractive evidence source, as it accumulates on undisturbed surfaces, often is overlooked by perpetrators, and contains sufficient human DNA for analysis. To assess whether SNPs genotyped from indoor dust using MPS could be used to detect known household occupants, 13 households were recruited and provided buccal samples from each occupant and dust from five predefined indoor locations. Thermo Fisher Scientific Precision ID Identity and Ancestry Panels were utilized for SNP genotyping, and sequencing was completed using Illumina^® chemistry. FastID, a software developed to permit mixture analysis and identity searching, was used to assess whether known occupants could be detected from associated household dust samples. A modified “subtraction” method was also used in FastID to estimate the percentage of alleles in each dust sample contributed by known and unknown occupants. On average, 72% of autosomal SNPs were recovered from dust samples. When using FastID, (a) 93% of known occupants were detected in at least one indoor dust sample and could not be excluded as contributors to the mixture, and (b) non-contributor alleles were detected in 54% of dust samples (29 ± 11 alleles per dust sample). Overall, this study highlights the potential of analyzing human DNA present in indoor dust to detect known household occupants, which could be valuable for investigative leads.     

Substitution of human for horse urine disproves an accusation of doping*

In order to detect switching and/or manipulation of samples, the owner of a stallion asked our lab to perform a DNA test on a positive doping urine sample. The objective was to compare the urine DNA profile versus blood and hair DNA profiles from the same stallion. At first, 10 microsatellite markers were investigated to determine the horse identity. No results were obtained when horse specific markers were typed in the urine sample. In order to confirm the species origin of this sample we analyzed the mitochondrial cytochrome b gene. This analysis from blood and hair samples produced reproducible and clear PCR-RFLP patterns and DNA sequence match with those expected for horse, while the urine sample results were coincident with human. These results allowed us to exclude the urine sample from the questioned stallion and determine its human species origin, confirming the manipulation of urine sample.     

An Accelerated Analytical Process for the Development of STR Profiles for Casework Samples

Significant efforts are being devoted to the development of methods enabling rapid generation of short tandem repeat (STR) profiles in order to reduce turnaround times for the delivery of human identification results from biological evidence. Some of the proposed solutions are still costly and low throughput. This study describes the optimization of an analytical process enabling the generation of complete STR profiles (single‐source or mixed profiles) for human identification in approximately 5 h. This accelerated process uses currently available reagents and standard laboratory equipment. It includes a 30‐min lysis step, a 27‐min DNA extraction using the Promega Maxwell^®16 System, DNA quantification in <1 h using the Qiagen Investigator^® Quantiplex HYres kit, fast amplification (<26 min) of the loci included in AmpF?STR^® Identifiler^®, and analysis of the profiles on the 3500‐series Genetic Analyzer. This combination of fast individual steps produces high‐quality profiling results and offers a cost‐effective alternative approach to rapid DNA analysis.     

常染色体21个SNPs多态性分型方法研究

目的建立常染色体21个SNPs的多态性分型方法。方法采用荧光标记公用引物和等位基因特异性引物原理设计SNP复合扩增引物体系,对45个备选SNP位点筛选,选出21个及性别Amelogenin构成复合扩增体系。PCR产物经3130XL型电泳仪电泳分离,GeneMaperTM3.0数据分析软件分析结果。同时随机选取6份样品,使用测序方法对SNP分型并进行测序验证。结果应用本研究建立的复合扩增体系扩增样品,产物经毛细管电泳后,每个SNPs均可正确判定基因型。随机选取6份样品SNPs位点测序结果显示,荧光标记SNPs复合扩增分型与直接测序结果完全一致。结论本研究建立的荧光标记公有引物特异性片段常染色体21个SNPs复合扩增方法是SNP多态性分析的一种有效方法,并有助于解决SNP分型识别能力、效率、通量和高成本的问题。     

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids, ,

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body‐fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR‐Duet^? kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand‐alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification.     

The Use of Mitochondrial DNA Single Nucleotide Polymorphisms to Assist in the Resolution of Three Challenging Forensic Cases

Abstract:  Mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) in an 11-plex assay were typed in three missing person cases involving highly degraded human remains. Unlike the traditional forensic approach to analyzing mtDNA which focuses on sequencing portions of the noncoding Control Region, this assay targets discriminatory SNPs that reside principally in the coding region. In two of the cases, the SNP typing successfully excluded one of two reference families that could not be excluded on the basis of mtDNA hypervariable region sequencing alone, and resulted in the final resolution of both decades-old cases. In a third case, SNP typing confirmed the sorting and reassociation of multiple commingled skeletal elements. The application of a specific mtDNA SNP assay in these cases demonstrates its utility in distinguishing samples when the most common Caucasian hypervariable region type is encountered in forensic casework.