MtSNP typing before mtDNA sequencing: Why do it?

Analysis of control mitochondrial DNA (mtDNA) hypervariable regions is sometimes the only available method to study hair evidence in forensic casework although being a laborious technique. Nowadays there is a huge interest in new genetic markers such as single nucleotide polymorphisms (SNPs) to type degraded forensic samples. For that purpose, a 10-Plex mitochondrial SNP for haplogroup typing, chosen from several SNP studies and useful to study the most common populations in our laboratory was applied in forensic casework. Hair shafts from three forensic cases with different ethnic backgrounds were studied with mtDNA sequencing and compared with mitochondrial SNPs (mtSNPs) study. Coding mtSNP typing prior to sequencing can allow for a rapid screening in forensic casework, which is emphasized in the first two cases. Moreover, in cases in which mtDNA sequencing fails, mtSNPs can still be detected. This 10 SNP loci multiplex provides a less expensive and simpler method for mitochondrial typing compared to control region mtDNA sequencing, especially when used as a fast screening method.     

Testing the performance of mtSNP minisequencing in forensic samples

There is a growing interest among forensic geneticists in developing efficient protocols for genotyping coding region mitochondrial DNA (mtDNA) SNPs (mtSNPs). Minisequencing is becoming a popular method for SNP genotyping, but it is still used by few forensic laboratories. In part, this is due to the lack of studies testing its efficiency and reproducibility when applied to real and complex forensic samples. Here we tested a minisequencing design that consists of 71 mtSNPs (in three multiplexes) that are diagnostic of known branches of the R0 phylogeny, in real forensic samples, including degraded bones and teeth, hair shafts, and serial dilutions. The fact that amplicons are short coupled with the natural efficiency of the minisequencing technique allow these assays to perform well with all the samples tested either degraded and/or those containing low DNA amount. We did not observe phylogenetic inconsistencies in the 71 mtSNP haplotypes generated, indicating that the technique is robust against potential artefacts that could arise from unintended contamination and/or spurious amplification of nuclear mtDNA pseudogenes (NUMTs).     

A DNA microarray system for forensic SNP analysis

Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future.     

Minisequencing-based genotyping of Duffy and ABO blood groups for forensic purposes

Duffy and ABO blood group genetic polymorphisms were studied by minisequencing analysis of single-nucleotide polymorphisms (SNPs) at nucleotide positions--33, 125, 265, and 298 of the Duffy gene and at nucleotide positions-261, 297, 467, 646, and 703 of the ABO gene. In an Italian population sample, we found four alleles and seven genotypes for the Duffy and six alleles and 16 genotypes for the ABO systems. The lower limit for reproducible results was 200 pg DNA, with a range of up to 10 ng and an optimum at 1 ng. All of the 16 analyzed inclusive paternity tests were also consistent with parentage and two out of four inconsistencies with parentage cases were excluded by one or more SNPs. Although Duffy and ABO SNP typing show lower informativeness than most current forensic tests, their robustness, the limited population distribution of FY* Fy type, and the sensitivity of the minisequencing technology suggest that these markers can be useful in selected forensic applications.     

Application of mtDNA SNP analysis in forensic casework

In this study six forensic cases are presented where the routine analysis of samples for short tandem repeats (STRs) failed. The sequencing of the mitochondrial hypervariable region I (HVR I) also failed. Nevertheless, it was possible to analyse the samples with mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) via SNaPshot technique. The age of the analysed samples ranged from 2 months to 1400 years. Saliva-, blood-, sperm-, hair-, tooth- and bone-samples were investigated. Furthermore the mtDNA SNP analysis of a forensic case sample showing a mixed stain profile is presented. It was possible to discriminate two different haplogroups in this mixed-person stain. If compared to another mtDNA SNP profile that was found in a hair, the discriminating SNPs of the hair were as well found in the mixed-person stain.To disburden the SNP analysis in forensic casework, haplogroup assignment criteria and quality criteria for mtDNA SNaPshot analysis are announced.     

Y-SNP-genotyping - a new approach in forensic analysis

Y-chromosomal DNA polymorphisms, especially Y-STRs are well established in forensic routine case work. The STRs are used for identification in paternity deficiency cases and stain analysis with complicate mixtures of male and female DNA. In contrast, Y-chromosomal SNPs are a new tool in forensic investigations. At present, Y-SNPs are mainly used in molecular anthropology for evolutionary studies. Nevertheless, these markers could also provide very useful information for the analysis of forensic cases. The aim of the presented study was to test Y-SNP-typing for stain analyses using different methods-SNaPshot and MALDI-TOF MS. Both methods are based on the principle of minisequencing. The selected Y-SNP markers are suited to define the most important European haplogroups.     

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.     

微测序技术检测12个Y—SNP及其遗传多态性

目的应用SNaPshotKit对Y染色体上12个SNP位点进行快速而准确的检测,对四川地区78个汉族男性无关个体进行群体遗传学研究,并对陈旧骨骼和性犯罪案件的相关物证进行检验。方法对SRY2627、SRY1532、M13、M20、SRY8299、Tat、M69及M9、92R7、M17、M19、M112两组共12个Y-SNP位点进行复合扩增,PCR产物经纯化处理后,采用SNaPshotKit试剂结合毛细管电泳技术对单核苷酸多态性进行检测。结果建立了12个Y-SNP位点的微测序快速检测系统,在四川地区人群中发现M9、SRY8299二个位点存在变异。结论复合扩增结合微测序技术能够同时对多个Y-SNP的多态性进行快速而准确的检测,建立的检测系统在法医学个体识别中具有应用价值。     

用dHPLC技术检测线粒体DNA编码区单核苷酸多态性

目的研究线粒体DNA(m tDNA)编码区单核苷酸多态性,建立检测m tDNA编码区单核苷酸多态性(SNP)的变性高效液相色谱(dHPLC)方法。方法设计针对线粒体DNA编码区nt10287-10679及nt8507-8805引物,应用dHPLC技术检测其序列多态性。结果100例中国汉族无关个体中,m tDNA nt10287-10679检出13个SNP位点,13种单倍型,基因多样性(H)为70.79%,偶合概率(P)为29.92%;m tDNA nt8507-8805检出10个SNP位点,12种单倍型,H为70.42%,P为30.28%;两段序列联合起来共检出23个SNP位点,23种单倍型,H为84.14%,P为16.70%。结论所建立的dHPLC方法可用于快速、准确地检测m tDNA编码区序列多态性;m tDNA编码区多态性位点作为m tDNA控制区多态性位点的补充,联合应用可以提高m tDNA的个体识别能力。     

Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing

The development of new methodologies for high-throughput SNP analysis is one of the most stimulating areas in genetic research. Here, we describe a rapid and robust assay to simultaneously genotype 17 mitochondrial DNA (mtDNA) coding region SNPs by minisequencing using SNaPshot. SNaPshot is a methodology based on a single base extension of an unlabeled oligonucleotide with labeled dideoxy terminators. The set of SNPs implemented in this multiplexed SNaPshot reaction allow us to allocate common mitochondrial West Eurasian haplotypes into their corresponding branch in the mtDNA skeleton, with special focus on those haplogroups lacking unambiguous diagnostic positions in the first and second hypervariable regions (HVS-I/II; by far, the most common segments analyzed by sequencing). Particularly interesting is the set of SNPs that subdivide haplogroup H; the most frequent haplogroup in Europe (40–50%) and one of the most poorly characterized phylogenetically in the HVS-I/II region. In addition, the polymorphic positions selected for this multiplex reaction increase considerably the discrimination power of current mitochondrial analysis in the forensic field and can also be used as a rapid screening tool prior to full sequencing analysis. The method has been validated in a sample of 266 individuals and shows high accuracy and robustness avoiding both the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the need for high quantities of DNA template.     

Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing

The development of new methodologies for high-throughput SNP analysis is one of the most stimulating areas in genetic research. Here, we describe a rapid and robust assay to simultaneously genotype 17 mitochondrial DNA (mtDNA) coding region SNPs by minisequencing using SNaPshot. SNaPshot is a methodology based on a single base extension of an unlabeled oligonucleotide with labeled dideoxy terminators. The set of SNPs implemented in this multiplexed SNaPshot reaction allow us to allocate common mitochondrial West Eurasian haplotypes into their corresponding branch in the mtDNA skeleton, with special focus on those haplogroups lacking unambiguous diagnostic positions in the first and second hypervariable regions (HVS-I/II; by far, the most common segments analyzed by sequencing). Particularly interesting is the set of SNPs that subdivide haplogroup H; the most frequent haplogroup in Europe (40-50%) and one of the most poorly characterized phylogenetically in the HVS-I/II region. In addition, the polymorphic positions selected for this multiplex reaction increase considerably the discrimination power of current mitochondrial analysis in the forensic field and can also be used as a rapid screening tool prior to full sequencing analysis. The method has been validated in a sample of 266 individuals and shows high accuracy and robustness avoiding both the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the need for high quantities of DNA template.     

Make it "SNPPY" - Updates to SRM 2391d: PCR-Based DNA Profiling Standard

Standard Reference Material (SRM) 2391d: PCR-Based DNA Profiling Standard was released to the forensic community in 2019. Next Generation Sequencing (NGS) was used as the primary method of certification, where certified values were assigned when a high coverage sequence string was available for a marker. Using NGS to assign values has allowed for additional marker sets beyond short tandem repeat (STR) loci, including single nucleotide polymorphisms (SNPs) and mitochondrial DNA (mtDNA) whole genome sequences, to be included in the Certificate of Analysis (COA). Since the 2019 release, several commercial NGS panels have become available including the Verogen ForenSeq mtDNA Control Region, mtDNA Whole Genome, MainstAY, and Kintelligence Kits. In addition, three community Ion AmpliSeq panels from Thermo Fisher (MH-74 Plex, VISAGE, and Y-SNP) are now available. While the mtDNA whole genome sequence for the components are already included and no new STR markers are introduced by MainstAY, the other recently released panels allow for the inclusion of > 11,000 additional SNPs (e.g., identity, ancestry, phenotype, kinship, and X- and Y-SNPs) and 74 microhaplotypes to the COA for SRM 2391d in an update completed by fall of 2022.     

Analysis of genetic polymorphisms associated with the presence of freckles for phenotypic prediction

The prediction of externally visible characteristics (EVCs) is a commonly used practice by the forensic sciences as an important resource in the investigation of criminal cases in which the identity of perpetrators or victims is unknown or even to recognize decomposed cadavers. With this purpose, genetic markers associated with pigmentation traits have been widely studied by forensic scientists and, nowadays, it is possible to predict phenotypic characteristics such as hair, eyes and skin colour, as well as the presence of skin freckles by analysing single nucleotide polymorphisms (SNPs). In this study, we analysed the association of six SNPs located in pigmentation genes to the presence of freckles in individuals from the Brazilian population for forensic DNA phenotyping. The study was based within the context of a larger project on a population sample of 534 adult Brazilians of both sexes and different skin colours. DNA was extracted from peripheral blood and genotyped using the TaqMan® OpenArray® Real-Time PCR System (ThermoFischer Scientific) technique. Statistical analyses were carried out with the R software (version 4.0.2). As for the results obtained, three SNPs were shown to be statistically associated to the freckling, rs12203592, rs1800404 and rs222847, with CT, AG and AA genotypes being the main contributors, respectively. Variables such as sex of the individuals and skin colour were found to also contribute to the manifestation of this pigmentation trait. Further statistical analyses will be carried out to evaluate the possibility of using the SNPs in this study for phenotyping prediction of the Brazilian population, improving existing DNA phenotyping models in forensic sciences.     

法医SNP复合检测体系的构建及应用

目的构建48-SNP位点复合检测体系,用于个体识别、性别鉴定、ABO基因分型。方法采集225份无关个体样本(血斑及口腔拭子),18份案例样本(不同组织及体液斑),选择43个常染色体位点、4个ABO基因位点和1个性别鉴定位点,根据单碱基延伸技术通过GenomeLabTMSNPstream基因分型系统进行SNP分型;并检测体系灵敏度、同一个体不同组织同一性及模拟腐败检材。结果 48-SNP体系分型结果与测序结果的一致性为100%,最小DNA检出量为0.25ng,不同组织来源样本检测同一性很好;利用该体系检测225名无关汉族个体,所有位点均符合Hardy-Weinberg平衡,整个系统的随机匹配概率为9.4×10-18,累积非父排除率(CEP)为0.999 788,累积个体识别率大于0.999 999 999 999 999 99。结论本文48-SNP体系能同时进行个体识别、ABO基因分型和性别鉴定,可以作为现有STR检验体系的补充。     

SNPs and MALDI-TOF MS: tools for DNA typing in forensic paternity testing and anthropology

DNA markers used for individual identification in forensic sciences are based on repeat sequences in nuclear DNA and the mitochondrial DNA hypervariable regions 1 and 2. An alternative to these markers is the use of single nucleotide polymorphisms (SNPs). These have a particular advantage in the analysis of degraded or poor samples, which are often all that is available in forensics or anthropology. In order to study the potential of SNP analysis in these fields, 41 SNPs were selected on the basis of following criteria: conservation, lack of phenotypic expression, and frequency of occurrence in populations. Thirty-six autosomal SNPs were used for genotyping 21 inclusionary and 3 exclusionary paternity cases. The behavior of 5 X-chromosome SNPs was analyzed in a French representative population. Our approach to SNP typing is a multiplex PCR based amplification followed by simultaneous detection by primer extension (PEX) analyzed by Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The selected autosomal SNPs showed independent inheritance and gave clear results in paternity investigation. All X-SNPs were useful as both paternity and identification markers. PEX and MALDI-TOF MS, with their high sensitivity, precision and speed, gave a powerful method for forensic and anthropological exploitation of biallelic markers.     

Multiplex PCR development of Y-chromosomal biallelic polymorphisms for forensic application

Single-nucleotide polymorphisms of Y chromosome (Y-SNPs) are a class of markers of interest in forensic investigations, because many of them show regional specificity, providing useful information about the geographic origin of a subject or evidence under investigation. A first multiplex with 7 SNPs (M35, M89, M9, M170, M172, M45, M173), which occur in the basal branches of the phylogenetic tree and are able to assign a subject to known most frequent European haplogroups, was designed. SNP genotyping was accomplished by hot-start PCR with primers amplifying fragments between 96 and 136 nucleotides, minisequencing, and capillary electrophoresis of extension products. Ninety seven subjects of known geographic provenance were studied, of which 68 from Europe. Of these, 57 had mutations found more frequently in European haplogroups and 11 more frequent in Asian populations. Subjects from non-European countries were also examined and had haplogroups common in their regions of provenance. Experiments with low molecular weight DNA gave positive amplification from 1 ng of DNA for all seven SNPs.     

Characterization of the Caucasian haplogroups present in the SWGDAM forensic mtDNA dataset for 1771 human control region sequences. Scientific Working Group on DNA Analysis Methods

Currently, the Scientific Working Group on DNA Analysis Methods (SWGDAM) mtDNA dataset is used to infer the relative rarity of mtDNA profiles (i.e., haplotypes) obtained from evidence samples and for identification of missing persons. The Caucasian haplogroup patterns in this forensic dataset have been characterized using phylogenetic methods. The assessment reveals that the dataset is relevant and representative of U.S. and European Caucasians. The comparisons carried out were both the observation of variable sites within the control region (CR) and the selection of a subset of these sites, which partition the variation within human mtDNA control region sequences into clusters (i.e., haplogroups). The aligned sequence matrix was analyzed to determine both single nucleotide polymorphisms (SNPs) in a phylogenetic context, as well as to check and standardize haplogroup designations with a focus on determining the characters that define these groups. To evaluate the dataset for forensic utility, the haplogroup identifications and frequencies were compared with those reported from other published studies.     

Accumulation of population polymorphisms in the mitonuclear genome with probable adaptive effect to extreme environments as altitude for forensic purposes

We analyzed the accumulation of population polymorphism in 2504 individuals - nuclear genomes (nDNA) of 26 populations (81 genes associated to extreme environments) and 3295 mitochondrial genomes (mtDNA) of 47 populations with the aim to found mitonuclear relationship associated an extremes environment as altitude. For that, we use an algorithm developed by us to determine the accumulation of polymorphisms by segments in the genome and thus be able to perform the multivariate analysis to found SNPs differences and similarities among populations. The results showed in Peruvian population a statistically significant mitonuclear relationship for 113/293970 nDNA SNPs in 16/81 genes. In the case of the mtDNA, we found a statistically significant mitonuclear relationship for 6/22 mtDNA positions – Gene. Additionally for the Peruvian population, the MRPP3 had the greatest polymorphism contribution with respect to other populations. Then, these nDNA and mtDNA SNPs in genetically close populations to Peru can be applied to forensic genomic phenotyping to identify groups likely adapted to extreme conditions (such as altitude) or make individualization between low and high altitude populations.     

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA

Six multiplex PCR systems using single‐base extension reactions to analyze 46 mitochondrial DNA (mtDNA)‐coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.