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.     

Analysis of mitochondrial SNPs in addition to conventional STR-typing in a case of aggravated theft

In the field of forensic DNA typing, the analysis of Short Tandem Repeats (STRs) can fail in cases of degraded DNA. The typing of coding region Single Nucleotide Polymorphisms (SNPs) of the mitochondrial genome provides an approach to acquire additional information. In the examined case of aggravated theft, both suspects could be excluded of having left the analyzed hair on the crime scene by SNP typing. This conclusion was not possible subsequent to STR typing. SNP typing of the trace on the torch light left on the crime scene increased the likelihood for suspect no. 2 to be the origin of this trace. This finding was already indicated by STR analysis. Suspect no. 1 was excluded for being the origin of this trace by SNP typing which was also indicated by STR analysis. A limiting factor for the analysis of SNPs is the maternal inheritance of mitochondrial DNA. Individualisation is not possible. In conclusion, it can be said that in the case of traces which cause problems with conventional STR typing the supplementary analysis of coding region SNPs from the mitochondrial genome is very reasonable and greatly contributes to the refinement of analysis methods in the field of forensic genetics.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Analysis of forensic samples in Banco Nacional de Datos Genéticos

Analysis of forensic samples to evaluate the rate of success for molecular markers: autosomal STRs, Y chromosome, and mitochondrial DNA. Since 2006 to date a total of 390 forensic samples were analyzed: bones, teeth, hairs, swabs, stains and paraffin embedded tissue. Bones and teeth, were pulverized in a Freezer Mill, extracted by chloroform/phenol/isoamyl alcohol method, and then purified with Centricon 100 columns. DNA from paraffin was extracted with QIAmp DNA Mini kit (QIAGEN). Mitochondrial DNA Control Region sequences were determined for regions HV1/HV2. Sequencing was performed using the BigDye^® Terminator v 1.1 Kit and analyzed in ABIPRISM^® 3100 Genetic Analyzer (AB). STRs were amplified using Amp FlSTR Identifiler^®, Minifiler^® and YFiler^® Kit (AB) and analyzed in ABI PRISM^® 3100 Genetic Analyzer and ABI PRISM^® 3130xl Genetic Analyzer (AB). Among forensic samples, bones and teeth analyzed for autosomal STRs, we obtained successful results in all of them. Incomplete typing are represented by loci of higher molecular weight, which demonstrates the poor quality of the sample due to its state of degradation and obtained better results using mini STRs. Successful results in sequencing for mitochondrial HV1 region for all samples analyzed, but in few hair samples we obtained mixed sequences and that represented important difficulties for the analysis. Age of samples and conservation are factors related which affect DNA viability. Autosomal STRs solved all the samples analyzed in our study, but Y chromosome analysis and mitochondrial DNA sequencing are also important and necessary markers in some forensic cases.     

Forensic Informativity of ~3000 bp of Coding Sequence of Domestic Dog mtDNA

The discriminatory power of the noncoding control region (CR) of domestic dog mitochondrial DNA alone is relatively low. The extent to which the discriminatory power could be increased by analyzing additional highly variable coding regions of the mitochondrial genome (mtGenome) was therefore investigated. Genetic variability across the mtGenome was evaluated by phylogenetic analysis, and the three most variable ~1 kb coding regions identified. We then sampled 100 Swedish dogs to represent breeds in accordance with their frequency in the Swedish population. A previously published dataset of 59 dog mtGenomes collected in the United States was also analyzed. Inclusion of the three coding regions increased the exclusion capacity considerably for the Swedish sample, from 0.920 for the CR alone to 0.964 for all four regions. The number of mtDNA types among all 159 dogs increased from 41 to 72, the four most frequent CR haplotypes being resolved into 22 different haplotypes.     

Multiplex DNA amplification and barcoding in a single reaction for 454 Roche sequencing: A comprehensive study on the control region of the mitochondrial genome

The analysis of the non-coding region of the mitochondrial genome using Sanger sequencing remains a laborious and time-consuming assay with too low resolution for the identification of low-frequency heteroplasmy or for mixture interpretation. In this study, an experimental design was tested in which the complete hypervariable region of the mitochondrial genome was sequenced using a novel barcoding strategy. The strategy involves a single-step multiplex nested PCR and we demonstrate its effectiveness by sequencing two multiplex reactions of two amplicons each covering the complete hypervariable region of the mitochondrial genome for 58 reference samples, 30 of which were analysed in triplicate, and 10 casework samples, each analysed in triplicate, on a 454 Roche DNA pyrosequencer with GS FLX chemistry using Multiplex Identifier (MID) primers to discriminate between samples. The generated reads for forensic (±3600 reads/MID) and reference samples (±466 reads/MID) allowed us to evaluate the accuracy in SNP calling and the variation in heteroplasmy and sequencing error rates in homopolymeric stretches between replicates.     

Mitochondrial profiling of dog hairs

In order to make greater use of dog hairs as forensic evidence, we have developed a robust method for duplex amplification of adjacent 306 and 332bp amplicons within the 5' hypervariable region (5' HVR) of the canine mitochondrial control region. In support of this, a 595bp region covering 35 polymorphic sites has been sequenced from the blood of 105 UK dogs. In total, 30 different haplotypes were observed, 13 only once whilst the commonest was seen 14 times; the overall exclusion capacity is 0.929. One animal was heteroplasmic in blood for a single base deletion and showed phenotypes ranging from near complete deletion to a predominance of the base among a sample of 12 hairs. In contrast, no evidence of heteroplasmy was seen in single hairs from 20 dogs which were not visibly heteroplasmic in blood. Phylogenetic analysis and comparisons with other published databases highlighted instances of possible recurrent mutation which may be relevant when interpreting single base differences between samples.     

Optimization and validation of 10 mitochondrial DNA SNPs using SNaPshot Kit

In some forensic cases, nuclear DNA is degraded and cannot be analyzed. In such a case mitochondrial DNA (mtDNA) is usually used in forensic cases for identification because of its special features as high number of copies per cell, maternal inheritance and high mutation rate. Single nucleotide polymorphisms (SNPs) represent the most abundant class of human polymorphisms. The aim of this study was optimization of 10 mtDNA SNPs by using SNaPshot minisequencing technique on ABI310 genetic analyser in forensic molecular genetics laboratory. At the end of this study, the optimization of minisequencing technique was done by changing some assay parameters. Also, during the optimization of 10 mtSNP loci in our laboratory.     

A multipurpose panel of microhaplotypes for casework

Microhaplotypes (MHs) are SNP-based multiallelic loci that have several advantages over individual SNPs and short tandem repeats (STRs). For several years we have been searching for better MHs based on the effective number of alleles at a locus (A_e) and the locus informativeness for population relationships (I_n) with thoughts of incorporating MHs into casework. We genotyped a multiplex of our best 90 MHs on 79 populations. We have ranked the 90 by A_e and analyzed the top 24 to evaluate their potential value in forensic casework. We chose 24 to compare with the popSTR dataset of 20 CODIS markers plus four other STRs commonly typed. PopSTR has full data on 32 populations; our 24 MHs have full data on 79 pops. We have compared the two sets of 24 loci (MH and STR) in four areas: individualization, biogeographic ancestry, kinship analysis, and mixture resolution.     

Assessing the potential application of X-chromosomal haploblocks in population genetics and forensic studies

Haploblocks are segments of the genome with little recombination that may be of interest in forensic and population genetics. Criteria to select autosomal haploblocks have been previously described, leading to the identification of candidate regions that, a priori, met the conditions to be used as forensic genetic markers. Still, the potential of X-chromosomal haploblocks remains unexplored.The present work aimed to provide basis for designing strategies for selection of X-haploblocks defined by single nucleotide polymorphisms (SNPs) using next generation sequencing approach. The potential application in population genetics and forensic studies was addressed. One of the conditions considered in the haploblock selection was the simultaneous inclusion of short tandem repeats (STRs) currently used in forensic casework to allow the distinction between SNP-defined haplotypes and increase the resolution for fine-scale studies. Given the size of the X chromosome (∼150 Mbps), only four haploblocks could be selected in order to guarantee their independence.     

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.     

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.     

A Three‐Locus,PCR‐based Method for Forensic Identification of Plant Material

Plant residue is currently an underutilized resource in forensic investigations despite the fact that many crime scenes, as well as suspects and victims, harbor plant‐derived residue that could be recovered and analyzed. Notwithstanding the considerable skill of forensic botanists, current methods of species determination could benefit from tools for DNA‐based species identification. However, DNA barcoding in plants has been hampered by sequence complications in the plant genome. Following a database search for usable barcodes, broad‐spectrum primers were designed and utilized to amplify and sequence the rbcL, trnL‐F, and rrn18 genetic loci from a variety of household plants. Once obtained, these DNA sequences were used to design species‐targeted primers that could successfully discriminate the source of plant residue from among the 21 species tested.