Human Identification Kit Research Articles

FORENSIC ODONTOLOGY SUSTAINABLE GOALS

The Importance of promoting Human Rights in the Recovery and Identification of Missing and Unidentified persons was presented at 9th International Dental Ethics and Law Society (IDEALS) congress in 2012 and was first introduced in May 2013 at the annual meeting of the Disaster Victim Identification (DVI) conference organized by Interpol in Lyon. Dr. Emilio Nuzzolese, participating odontologist of the Interpol Forensic Odontology working group, highlighted the need to apply the highest identification standards and Interpol guidelines to all unidentified human remains, equally in a DVI scenario as well as in a single missing and unidentified person. In 2015 these concepts also inspired the creation of an international group of odontologists from twelve Countries called ‘Forensic Odontology for Human Rights’. The group began promoting the need to protect the human rights of the dead to have a name and an identity by participating in conferences and meetings, and through publications and training at most international forensic events. The mentoring of colleagues with the goal of promoting an authentic involvement of forensic odontology in the identification process was also instigated. The continuous growth of the group, along with the need to involve forensic experts from other disciplines, led to the constitution of the Association Forensic Odontology for Human Rights (AFOHR) and the inauguration of humanitarian forensic odontology. Having become a recognized voluntary organization in the context of humanitarian forensics, in 2019 AFOHR board members discussed the steps needed to achieve the rights of having a date of birth and an identity for every deceased individual. Eight forensic odontology sustainable goals were approved: human rights of the dead; human identification action; international partnership; human forensic odontology; strong forensic odontology; human identification kit; quality age determination; dental evidence in crimes against vulnerable persons. Under the auspices of AFOHR that aim is to inspire colleagues and forensic associations to the best administration of Justice for best practices in human identification and age estimation.

Population genetic data for 17 non-CODIS STR loci for the Saudi Arabian population using the SureID®23comp Human Identification Kit

Our previous work focused on validation the SureID 23comp Human Identification Kit (Health Gene Technologies, China), following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples from the population of Saudi Arabia. The kit genotypes 22 STRs, 17 of which are non-CODIS, and Amelogenin. The validation tests showed that it has the potential to increase the power of testing in complex cases.In this paper, the allele frequency data, common forensic parameters for the 17 non-CODIS STR loci are presented. We found the majority of loci had an excess of homozygosity in the data set, which is most likely explained by the relatively high levels of consanguinity in the population of Saudi Arabia.

Utility of Circulating Cell-Free DNA in Assessing Microsatellite Instability and Loss of Heterozygosity in Breast Cancer Using Human Identification Approach.

The diagnostic and prognostic utility of circulating cell-free DNA (cfDNA) in breast cancer (BC) patients was recently reported. Here, we investigated the use of cfDNA to examine microsatellite instability (MSI) and loss of heterozygosity (LOH) for early BC diagnosis. cfDNA and genomic DNA from 41 female BC patients and 40 healthy controls were quantified using NanoDrop spectrophotometry and real-time PCR. The stability of genomic and cfDNA was assessed using a high-resolution AmpFlSTR MiniFiler human identification kit. Significant increases in cfDNA plasma concentrations were observed in BC patients compared to controls. The genotype distribution of the eight autosomal short tandem repeat (STR) loci D7S820, D13S317, D21S11, D2S1338, D18S51, D16S539, FGA, and CSF1PO were in Hardy–Weinberg equilibrium. Significant differences in the allele frequencies of D7S820 allele-8, D21S11 allele-29, allele-30.2, allele-32.2, and CSF1PO allele-11 were seen between BC patients and controls. LOH and MSI were detected in 36.6% of the cfDNA of patients compared to genomic DNA. This study highlights the utility of plasma-derived cfDNA for earlier, less invasive, and cost-effective cancer diagnosis and molecular stratification. It also highlights the potential value of cfDNA in molecular profiling and biomarkers discovery in precision and forensic medicine.

Evaluation of the InnoTyper21® system for the applications into trace and degraded DNA in the Korean population.

The InnoTyper 21® Human Identification kit consists of amelogenin and 20 bi-allelic Alus, retrotransposon markers existing abundantly in human genome. The InnoTyper 21® kit produces shorter amplicons (60-125 bp) than conventional short tandem repeat (STR) genotyping kit, then it is effective on the analysis of challengeable forensic samples including insufficient or highly degraded DNAs. Also, as the genotyping with InnoTyper21® kit is compatible with PCR and capillary electrophoresis, it is easy to incorporate into the workflow in forensic laboratories. In the internal validation of InnoTyper21® kit on sensitivity, degradation, and mixture studies for the evaluation in this study, we acquired full profiles on analyzing small concentration DNA (as low as 25 pg) and highly degraded DNA (up to 105 degradation index value). Through the Korean population study, forensic statistical parameters were investigated and a specific variant of T insertion in NBC51 was confirmed in six samples. Comparison of Korean population with five populations or 1000 Genomes Project data show Korean specific substructure. It is expected that the InnoTyper 21® kit will be used into the actual forensic cases, utilizing the population study investigated through this research.

Dog-bite-related attacks: A new forensic approach

Dog attacks today represent a health hazard considering that prevention strategies have not always been successful. The identification of the dog that attacked the victim is necessary, considering the civil or criminal consequences for the animal’s owner. An accurate scene analysis must be performed collecting a series of important information.Forensic investigations in dog attacks involve different methods, such as the evaluating of the canine Short Tandem Repeat (STR) typing in saliva traces on wounds or bite mark analysis, however, these techniques cannot always be applied. The effort to find new methods to identify the dog that attacked the victim represents a very interesting field for the forensic community.This study aims to propose an innovative approach, based on the identification of the victim's profile in the dog's mouth, using a buccal swab on the suspected aggressor dog, to find the victim’s genetic profile. In addition, a further goal of this study is to determine the persistence time of hexogen DNA in the dog’s mouth to define a timeframe for performing this particular technique.For this purpose, ten different dogs were used to aggressively bite a bovine sample (reference sample) to simulate the victim. For each dog two buccal swabs were taken at different time intervals: 30′, 45′, 60′, 90′, 120′, 150′, 180′ and 240′. The typing of the swabs provided an interpretable profile after 45′ while traces of bovine profile were found until 150′ after the dog attack simulation.These results could be improved using the human identification kit, which is more sensitive. In the light of this experimental study, the forensic community should consider using this approach in real casework studies with the aim of collecting new data, validating this technique for forensic use.

An evaluation of the SureID 23comp Human Identification Kit for kinship testing

Short tandem repeat (STR) profiling has been routinely used in kinship testing since the introduction of commercial kits in the mid-1990s. While 15 to 23 STR loci normally give definitive results in simple kinship testing, additional loci are sometimes required to resolve complex cases. The SureID 23comp Human Identification Kit, recently released by Health Gene Technologies (China), multiplexes amelogenin and 22 autosomal STRs, 17 of which are non-CODIS STRs. This enables the profiling of 38–40 loci when used in conjunction with widely used commercial kits. In this study, the kit was evaluated for kinship applications as a supplementary STR kit following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples. Performance was comparable with other commercial kits demonstrating: repeatability and reproducibility; precision (maximum s.d. 0.1048 nt); accuracy, all alleles were within ±0.41 nt compared to the actual sizes; heterozygous peak balances at all loci >68%; stutter ratios ranged from 3.8% to 16.15%; full profiles were generated with 125 pg DNA (95.12% of alleles at 62 pg),; and we found 100% concordance over 5 common STRs with the GlobalFiler kit.

Forensic statistical parameters of 22 autosomal STRs in Mestizos from the Peninsula of Yucatán, México

The Mestizo (admixed) population of the Peninsula of Yucatan (Southeast, México) characterizes by a predominant Native American origin involving the Mayan culture. Autosomal Short tandem repeats (STRs) are the election tool for human identification (HID) purposes, such as forensic casework and paternity testing, among others. Although there are autosomal STR databases for some Mexican populations, the HID kits with more STRs have poorly studied in this country. Therefore, we estimated forensic parameters for 22 autosomal STRs belonging to the PowerPlex® Fusion System Promega kit (Promega, Corp.) in a population sample of 733 Mexican Mestizos subjects from the Peninsula of Yucatán (Southeast, Mexico). Buccal swabs were used as DNA sample for direct PCR multiplex amplification for genotyping PowerPlex® Fusion System Promega kit. Forensic parameters were obtained with Powerstats 1.2 software. Hardy-Weinberg expectations (HWE) were estimated with GDA ver 1.1 software. Gene diversity was estimated with Shannon-Wiener (H`) and statistical differences with t-test modified by Hutcheson using Biodiv 5.1 software. Interpopulation comparisons were analyzed with Dice and Rogers-Tanimoto indexes and classification method of Unweighted Pair-Group Method (UPGMA). Genotype distribution was in agreement with HWE (p > 0.001), except for D1S1656. FGA and Penta E were the most polymorphic loci with 32 and 25 different alleles, respectively. Heterozygosity ranged from 0.6112 for D22S1045 to 0.9141 for D1S1656. The most discriminating loci were Penta E (0.9852) and FGA (0.9756). The combined power of discrimination and combined power of exclusion were 1.9093 × 10−26 and 99.9999833%, respectively. Gene diversity ranged from 1.21 for D22S1045 to 2.68 for Penta E. Inter-comparison populations did not show significant differences for gene diversity based on allelic frequencies of 22 studied autosomal STR. Our results provide a STR population dataset that allow confident interpretation of paternity tests and criminal cases carried out with this HID system in the Peninsula of Yucatan.

Inference of maternal uniparental disomy of the entire chromosome 2 from a paternity test.

Atypical situations arise during the constant resolution of paternity cases, which constitute challenges requiring additional genetic systems and non-standard methods. We report a paternity case presenting three alleged father (AF)-child incompatibilities for the markers TPOX, D2S441, and the indel locus B02 (11/11 vs 8/8; 14/14 vs 10/10; 2/2 vs1/1, respectively). Considering the presence of mutations/null alleles, the residual paternity indexes (PI) obtained with 23 autosomal short tandem repeats (STRs) and 38 indels suggest that the AF is the father (PI = 1.94e+011). Although the presence of few incompatibilities also could imply paternity of the AF brother, this hypothesis was less probable (PI = 3.20e+9) (W = 98.4 vs 1.6%, respectively). The inclusion of 23 Y-STR loci confirmed the paternity relationship in this case (global PI = 6.08e+15). However, the two multistep STRs and one indel incompatibilities allow discarding the mutation possibility. On the other hand, the confirmation of the homozygous STR genotypes with two different human identification kits and the low probability to find three null alleles (3.10e-8) allow rejecting the null allele presence hypothesis. Conversely, the child's homozygous genotype for maternal alleles in four markers located in the p and q arms of the chromosome 2 (TPOX, D2S441, D2S1338, and B02) suggests that maternal uniparental isodisomy better explains the relationship despite the presence of three paternal incompatibilities. In brief, when multiple incompatibilities are observed in paternity testing, the chromosomal location of the excluding loci and the use of additional genetic systems can be crucial to get confident kinship conclusions.

Paternity tests in Mexico: Results obtained in 3005 cases

National and international reports regarding the paternity testing activity scarcely include information from Mexico and other Latin American countries. Therefore, we report different results from the analysis of 3005 paternity cases analyzed during a period of five years in a Mexican paternity testing laboratory. Motherless tests were the most frequent (77.27%), followed by trio cases (20.70%); the remaining 2.04% included different cases of kinship reconstruction. The paternity exclusion rate was 29.58%, higher but into the range reported by the American Association of Blood Banks (average 24.12%). We detected 65 mutations, most of them involving one-step (93.8% and the remaining were two-step mutations (6.2%) thus, we were able to estimate the paternal mutation rate for 17 different STR loci: 0.0018 (95% CI 0.0005–0.0047). Five triallelic patterns and 12 suspected null alleles were detected during this period; however, re-amplification of these samples with a different Human Identification (HID) kit confirmed the homozygous genotypes, which suggests that most of these exclusions actually are one-step mutations. HID kits with ≥20 STRs detected more exclusions, diminishing the rate of inconclusive results with isolated exclusions (<3 loci), and leading to higher paternity indexes (PI). However, the Powerplex 21 kit (20 STRs) and Powerplex Fusion kit (22 STRs) offered similar PI (p = 0.379) and average number of exclusions (PE) (p = 0.339) when a daughter was involved in motherless tests. In brief, besides to report forensic parameters from paternity tests in Mexico, results describe improvements to solve motherless paternity tests using HID kits with ≥20 STRs instead of one including 15 STRs.

Analysis of fortuitous matches in a STR genotype database from Mexico and its forensic efficiency parameters

BackgroundCommercial kits for Human Identification (HID) purposes, based on 15 short tandem repeat (STR) regions, allow the resolution of most forensic and paternity cases. However, some pitfalls arise in situations such as identification of missing persons, disaster victims, and in motherless paternities. We describe nine fortuitous matches found during the inclusion of new DNA profiles and/or searching for missing persons within a Mexican STR database (N = 2000). For these cases, we estimated both the likelihood ratio (LR) and the modified LR according to National Research Council (NRC) recommendations (i.e., LRNRC).ResultsIn most cases, low LRNRC values (~1 or <1) and elevated presence of modal STR alleles (>50% loci) were indicative of fortuitous matches. We estimated a significant increase in the observed frequency of fortuitous matches in this database when using the PowerPlex 16 DNA-typing system. In addition, forensic efficiency parameters for this HID kit are reported for the State of Mexico based on a population sample selected from the STR database (n = 493). We describe the population structure and relationships, which detail comparative homogeneity among Mexican-Mestizos from the Central region, excluding the Western region.ConclusionsThe results suggest that additional / more powerful HID systems should be used when searching for relatives within STR databases in order to enable definitive conclusions, which are also required in cases of motherless paternity and massive disasters. The genetic structure described herein allows forensic geneticists to use alternative STR population data during statistical interpretation, given that most Mexican populations do not have their own STR databases.

Identification of a common single nucleotide polymorphism at the primer binding site of D2S1360 that causes heterozygote peak imbalance when using the Investigator HDplex Kit.

Phenomena known as null alleles and peak imbalance can occur because of mutations in the primer binding sites used for DNA typing. In these cases, an accurate statistical evaluation of DNA typing is difficult. The estimated likelihood ratio is incorrectly calculated because of the null allele and allele dropout caused by mutation-induced peak imbalance. Although a number of studies have attempted to uncover examples of these phenomena, few reports are available on the human identification kit manufactured by Qiagen. In this study, 196 Japanese individuals who were heterozygous at D2S1360 were genotyped using an Investigator HDplex Kit with optimal amounts of DNA. A peak imbalance was frequently observed at the D2S1360 locus. We performed a sequencing analysis of the area surrounding the D2S1360 repeat motif to identify the cause for peak imbalance. A point mutation (G>A transition) 136 nucleotides upstream from the D2S1360 repeat motif was discovered in a number of samples. The allele frequency of the mutation was 0.0566 in the Japanese population. Therefore, human identification or kinship testing using the Investigator HDplex Kit requires caution because of the higher frequency of single nucleotide polymorphisms at the primer binding site of D2S1360 locus in the Japanese population.

Estimating allele dropout probabilities by logistic regression: Assessments using Applied Biosystems 3500xL and 3130xl Genetic Analyzers with various commercially available human identification kits.

Phenomena called allele dropouts are often observed in crime stain profiles. Allele dropouts are generated because one of a pair of heterozygous alleles is underrepresented by stochastic influences and is indicated by a low peak detection threshold. Therefore, it is important that such risks are statistically evaluated. In recent years, attempts to interpret allele dropout probabilities by logistic regression using the information on peak heights have been reported. However, these previous studies are limited to the use of a human identification kit and fragment analyzer. In the present study, we calculated allele dropout probabilities by logistic regression using contemporary capillary electrophoresis instruments, 3500xL Genetic Analyzer and 3130xl Genetic Analyzer with various commercially available human identification kits such as AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. Furthermore, the differences in logistic curves between peak detection thresholds using analytical threshold (AT) and values recommended by the manufacturer were compared. The standard logistic curves for calculating allele dropout probabilities from the peak height of sister alleles were characterized. The present study confirmed that ATs were lower than the values recommended by the manufacturer in human identification kits; therefore, it is possible to reduce allele dropout probabilities and obtain more information using AT as the peak detection threshold.

A method to determine the 5′ end of the binding site of primers included in a commercially available forensic human identification kit

Analysis for short tandem repeat (STR) loci is widely performed in forensic laboratories for human identification that utilizes commercially available kits that employ fluorescently labeled primers and capillary electrophoresis. With only a few exceptions, the sequences of the primers included in a kit are not disclosed by the kit manufacturers. Therefore, we developed a simple method to determine the 5' end of the binding site of the primers included in commercial kits. Our method requires only custom primers and human genome sequence data and routinely used equipment and consumables. One or two custom primers are added to the PCR reaction mixture containing kit primers and input human DNA prior to amplification, and PCR products are separated by capillary electrophoresis after amplification. With this method we can determine which primer of the pair is fluorescently labeled and the 5' end of the binding site of primers based on the changes in an electropherogram that are caused by the addition of the custom primer(s), and the human genome sequence data. This method is also useful for the determination of the shortest possible lengths of labeled kit primers.

Concordance Study between the AmpFlSTR® SGM Plus™ and PowerPlex® 16 System Human Identification Kits in Bangladeshi Population

In this study, we are reporting the concordance study between profiles from AmpFlSTR ® SGM plus™ and that of PowerPlex®16 system kits in Bangladeshi Bangali population. DNA samples from 234 unrelated Bangladeshi Bangali individuals were evaluated employing both the SGM plus™ and PowerPlex ® 16 system human identification kits. Complete consistency was observed in 233 out of 234 profiles which represents substantial degree of concordance amounting 99.57% between these two kits. Only one discrepancy encompassing the dropout of allele 12 at D16S539 loci in PowerPlex ® 16 system was observed. Sequence analysis revealed two mutational events; one at 16 bp distal and another next to the 3' end of the PowerPlex 16 reverse primer of D16S539 locus. This study has shown the significance of using of alternative set of primer to address the false homozygosity issue as well as of bringing attention regarding the fact of discrepant typing from different sets of primers to the scientific community during database handling and identity or kinship investigations. and Santals, mainly labourers in the tea garden at Sylhet district. The Mongolian group is confined to the hills along the southern spur of the Shilong plateau, in Chittagong hill tracts and Madhupur forest. Chakam, Tripura, Garo, Murang and Moghs are the major Mongolid tribes in Bangladesh. The individuals recruited in this study belongs to only the mainstream Bangali population.

Kinship and Y-Chromosome Analysis of 7th Century Human Remains: Novel DNA Extraction and Typing Procedure for Ancient Material

To develop novel DNA extraction and typing procedure for DNA identification of the 7th century human remains, determine the familiar relationship between the individuals, estimate the Y-chromosome haplogroup, and compare the Y-chromosome haplotype with the contemporary populations. DNA from preserved femur samples was extracted using the modified silica-based extraction technique. Polymerase chain reaction amplification was performed using human identification kits MiniFiler, Identifiler, and Y-filer and also laboratory-developed and validated Y-chromosome short tandem repeat (STR) pentaplexes with short amplicons. For 244A, 244B, 244C samples, full autosomal DNA profiles (15 STR markers and Amelogenin) and for 244D, 244E, 244F samples, MiniFiler profiles were produced. Y-chromosome haplotypes consisting of up to 24 STR markers were determined and used to predict the Y-chromosome haplogroups and compare the resulting haplotypes with the current population. Samples 244A, 244B, 244C, and 244D belong to Y-chromosome haplogroup R1b and the samples 244E and 244F to haplogroup G2a. Comparison of ancient haplotypes with the current population yielded numerous close matches with genetic distance below 2. Application of forensic genetics in archaeology enables retrieving new types of information and helps in data interpretation. The number of successfully typed autosomal and Y-STR loci from ancient specimens in this study is one of the largest published so far for aged samples.

Validation of SRY Marker for Forensic Casework Analysis

Determining the gender of the source of forensic DNA evidence is based on the amelogenin test. However, at times the assay may not be indicative of gender assignment, because of deletions at the amelogenin site. Previously, we described successful coamplification of a marker residing within the SRY gene with the short tandem repeat markers from two commercially available human identification kits. The study herein addresses the validation of primers for the target SRY gene regarding specificity, sensitivity, and robustness. Among 115 unrelated male Slovenians no null allele was observed. Repeatable and reliable results were obtained from as little as 25 pg of template DNA, indicating a high sensitivity of detection for the assay. No polymerase chain reaction product was observed even at a concentration of 10 ng/microL of template female DNA. Additionally, the male specific marker could be detected in mixed male and female samples down to a ratio of 1:16.