Statistical Detection of Relatives Typed with Disjoint Forensic and Biomedical Loci.

In familial searching in forensic genetics, a query DNA profile is tested against a database to determine whether it represents a relative of a database entrant. We examine the potential for using linkage disequilibrium to identify pairs of profiles as belonging to relatives when the query and database rely on nonoverlapping genetic markers. Considering data on individuals genotyped with both microsatellites used in forensic applications and genome-wide SNPs, we find that ∼30%-32% of parent-offspring pairs and ∼35%-36% of sib pairs can be identified from the SNPs of one member of the pair and the microsatellites of the other. The method suggests the possibility of performing familial searches of microsatellite databases using query SNP profiles, or vice versa. It also reveals that privacy concerns arising from computations across multiple databases that share no genetic markers in common entail risks, not only for database entrants, but for their close relatives as well.

Sequenced-based Rwanda population provides insights into demographic history.

Rwanda is known as the heart of Africa, reflecting the history of the world. Colonization and genocide have led to Rwanda's existing genetic structure. Herein, we used massively parallel sequencing to analyze 296 loci in 185 Rwandans and constructed a database for Rwandan forensic data for the first time. We found the following results: First, forensic parameters demonstrated that all loci were highly informative and could be used for forensic identification and paternity tests in Rwandans. Second, we found that the differences in genetic background between Rwandans and other African populations were similar but slight, as indicated by the massively parallel sequencing panel. Rwandans belonged to the African population and were inseparable from populations from neighboring countries. Also, Rwandans were closer to the European and American populations because of colonization, war, and other reasons. There was no scientific basis for racial classification established by colonization. Further research still needs to be carried out on more loci and larger Rwandan samples.

MC1R and age heteroclassification of face phenotypes in the Rio Grande do Sul population.

BACKGROUND: Forensic DNA phenotyping (FDP) consists of the use of methodologies for predicting externally visible characteristics (EVCs) from the genetic material of biological samples found in crime scenes and has proven to be a promising tool in aiding human identification in police activities. Currently, methods based on multiplex assays and statistical models of prediction of EVCs related to hair, skin, and iris pigmentation using panels of SNP and INDEL biomarkers have already been developed and validated by the forensic scientific community. As well as traces of pigmentation, an individual's perceived age (PA) can also be considered an EVC and its estimation in unknown individuals can be useful for the progress of investigations. Liu and colleagues (2016) were pioneers in evidencing that, in addition to lifestyle and environmental factors, the presence of SNP and INDEL variants in the MC1R gene - which encodes a transmembrane receptor responsible for regulating melanin production - seems to contribute to an individual's PA. The group highlighted the association between these MC1R gene polymorphisms and the PA in the European population, where carriers of risk haplotypes appeared to be up to 2 years older in comparison to their chronological age (CA). PURPOSE: Understanding that genotype-phenotype relationships cannot be extrapolated between different population groups, this study aimed to test this hypothesis and verify the applicability of this variant panel in the Rio Grande do Sul admixed population. METHODS: Based on genomic data from a sample of 261 volunteers representative of gaucho population and using a multiple linear regression (MLR) model, our group was able to verify a significant association among nine intronic variants in loci adjacent to MC1R (e.g., AFG3L1P, TUBB3, FANCA) and facial age appearance, whose PA was defined after age heteroclassification of standard frontal face images through 11 assessors. RESULTS: Different from that observed in European populations, our results show that the presence of effect alleles (R) of the selected variants in our sample influenced both younger and older face phenotypes. The influence of each variant on PA is expressed as ß values. CONCLUSIONS: There are important molecular mechanisms behind the effects of MC1R locus on PA, and the genomic background of each population seems to be crucial to determine this influence.

Developmental validation of the STRSeqTyper122 kit for massively parallel sequencing of forensic STRs.

Massively parallel sequencing allows for integrated genotyping of different types of forensic markers, which reduces DNA consumption, simplifies experimental processes, and provides additional sequence-based genetic information. The STRseqTyper122 kit genotypes 63 autosomal STRs, 16 X-STRs, 42 Y-STRs, and the Amelogenin locus. Amplicon sizes of 117 loci were below 300 bp. In this study, MiSeq FGx sequencing metrics for STRseqTyper122 were presented. The genotyping accuracy of this kit was examined by comparing to certified genotypes of NIST standard reference materials and results from five capillary electrophoresis-based kits. The sensitivity of STRseqTyper122 reached 125 pg, and > 80% of the loci were correctly called with 62.5 pg and 31.25 pg input genomic DNA. Repeatability, species specificity, and tolerance for DNA degradation and PCR inhibitors of this kit were also evaluated. STRseqTyper122 demonstrated reliable performance with routine case-work samples and provided a powerful tool for forensic applications.

Pedigrees and Perpetrators: Uses of DNA and Genealogy in Forensic Investigations.

In the past few years, cases with DNA evidence that could not be solved with direct matches in DNA databases have benefited from comparing single-nucleotide polymorphism data with private and public genomic databases. Using a combination of genome comparisons and traditional genealogical research, investigators can triangulate distant relatives to the contributor of DNA data from a crime scene, ultimately identifying perpetrators of violent crimes. This approach has also been successful in identifying unknown deceased persons and perpetrators of lesser crimes. Such advances are bringing into focus ethical questions on how much access to DNA databases should be granted to law enforcement and how best to empower public genome contributors with control over their data. The necessary policies will take time to develop but can be informed by reflection on the familial searching policies developed for searches of the federal DNA database and considerations of the anonymity and privacy interests of civilians.

Evaluation of face masks as a valuable forensic DNA evidence in the post-COVID era.

After the onset of COVID-19 pandemic, a sharp surge in the usage of the face-masks throughout the globe has been observed. Pre-experiment survey of 252 individuals indicated a higher use of cotton-make masks (41%), followed by N-95 make (31%), and surgical disposable masks (26%). It was also further revealed that a higher fraction of individuals wear a face-mask more than 3 times (37%) before its disposal. In order to assess the potential usability of different mask types as forensic DNA evidence, a study was conducted on 50 healthy individuals. DNA content of different fractions such as the portion of mask covering the mouth region and the ear-piece showed a good source of host DNA. Though no statistically significant difference (P < 0.05) was found in the DNA quantity obtained from different face mask types, an increasing trend was obtained in the order: cloth make type (7.031 ± 0.31 ng), N-95 make (4.711 ± 0.15 ng), and surgical disposable type (2.17 ± 0.13 ng). The time of wearing of a face-mask showed a positive correlation with the yield of DNA irrespective of the face-mask type used. Samples retrieved from both the portions covering the mouth area and the ear-piece showed a good source of genomic DNA yielding an average of 4.82 ± 0.11 ng and 4.44 ± 0.10 ng of DNA, respectively. Irrespective of the face-mask types, number of reuse, and the portion of the mask, 66.66-96.11% of samples showed a complete autosomal STR DNA profile. This suggests that if a face-mask is found at the crime scene, it should be collected and preserved as a potential source of DNA evidence for routine forensic DNA analysis.

A genetic Study of the Ghanaian Population Using 15 Autosomal STR Loci.

Autosomal short tandem repeat (STR) population data collected from a well characterized population are needed to correctly assigning the weight of DNA profiles in the courtroom and widely used for ancestral analyses. In this study, allele frequencies for the 15 autosomal short tandem repeat (STR) loci included in the AmpFlSTR® Identifiler® plus kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818, FGA) were obtained by genotyping 332 unrelated individuals of Ghanaian origin. Statistical tests on STR genotype data showed no significant departure from Hardy-Weinberg equilibrium (HWE). The overall match probability, combined power of exclusion and combined power of discrimination for these loci were 1 in 3.85 × 1017, 0.99999893 and 0.99999998, respectively. Polymorphic information content (PIC) greater than 0.70 was observed for all loci except TH01 and D13S317. These statistical parameters confirm that this combination of loci is valuable for forensic identification and parentage analysis. Our results were also compared with those for 20 other human populations analyzed for the same set of markers. We observed that the Ghanaian population grouped with other African populations in two-dimensional principal coordinate (PCO) and a neighbor-joining (N-J) data mapping and placed closest to Nigerians. This observation reflects cultural similarities and geographical factors, coupled with the long history of migration and trading activities between Ghana and Nigeria. Our report provides what we believe to be the first published autosomal STR data for the general Ghanaian population using 15 loci genotyped using the AmpFlSTR® Identifiler® plus kit methodology. Our data show that the loci tested have sufficient power to be used reliably for DNA profiling in forensic casework and help to elucidate the genetic history of people living in the country.

Detecting DNA damage in stored blood samples.

Several commercially available quantitative real-time PCR (qPCR) systems enable highly sensitive detection of human DNA and provide a degradation index (DI) to assess DNA quality. From routine casework in forensic genetics, it was observed that DNA degradation in forensic samples such as blood samples stored under sub-optimal conditions leads to visible effects in multiplex analyses of short tandem repeat markers (STRs) due to decreased amplification efficiencies in longer amplicons. It was further noticed that degradation indices often remain below the value that is considered to be critical. Thus, the aim of this work was to systematically analyze this effect and to compare conventional qPCR assays with a modified qPCR approach using uracil DNA glycosylase (UNG) and DNA quality assessment methods based on electrophoresis. Blood samples were stored at three different storage temperatures for up to 316 days. Significantly increased DNA recovery was observed from samples stored at high temperatures (37 °C) compared samples stored at room temperature and 4 °C. We observed typical effects of degradation in STR analyses but no correlation between DI and storage time in any of the storage conditions. Adding UNG slightly increased the sensitivity of detecting DNA degradation in one of the qPCR kits used in this study. This observation was not confirmed when using a second qPCR system. Electrophoretic systems did also not reveal significant correlations between integrity values and time. Methods for detecting DNA degradation are usually limited to the detection of DNA fragmentation, and we conclude that degradation affecting forensic STR typing is more complex.

Evaluation of loci to predict ear morphology using two SNaPshot assays.

Human ear morphology prediction with SNP-based genotypes is growing in forensic DNA phenotyping and is scarcely explored in Pakistan as a part of EVCs (externally visible characteristics). The ear morphology prediction assays with 21 SNPs were assessed for their potential utility in forensic identification of population. The SNaPshot™ multiplex chemistries, capillary electrophoresis methods and GeneMapper™ software were used for obtaining genotypic data. A total of 33 ear phenotypes were categorized with digital photographs of 300 volunteers. SHEsis software was applied to make LD plot. Ordinal and multinomial logistic regression was implemented for association testing. Multinomial logistic regression was executed to construct the prediction model in 90% training and 10% testing subjects. Several influential SNPs for ear phenotypic variation were found in association testing. The model based on genetic markers predicted ear phenotypes with moderate to good predictive accuracies demonstrated with the area under curve (AUC), sensitivity and specificity of predicted phenotypes. As an additional EVC, the estimated ear phenotypic profiles have the possibility of determining the human ear morphology differences in unknown biological samples found in crimes that do not result in a criminal database hit. Furthermore, this can help in facial reconstruction and act as an investigational lead.

Development and validation of a new multiplex Y-STR panel designed to increase the power of discrimination.

In an attempt to increase the discrimination capacity (DC) and reduce the adventitious match probability, a 6-dye multiplex Y-chromosomal short tandem repeat (Y-STR) panel named Y34plex was constructed that combined 25 Y-chromosomal markers (DYS456, DYS627, DYS390, DYS570, DYS635, DYS385a/b, DYS448, DYS437, DYS533, DYS449, DYS481, DYS392, DYS391, DYS389I, DYS460, YGATAH4, DYS438, DYS389II, DYS19, DYS458, DYF387S1a/b, DYS439, DYS393, DYS576, and DYS518) in widely used commercial kits, with nine highly polymorphic Y-STR loci (DYS557, DYS527a/b, DYS593, DYS444, DYS596, DYS643, DYS447, DYS549, and DYS645). The Y34plex is a promising type system to distinguish both unrelated and related male individuals due to the incorporation of rapidly mutated Y-STR loci. A validation study of the Y34plex was performed and followed the guidelines of the Scientific Working Group on DNA analysis methods. Results show that full Y-STR profiles were obtained from male/female DNA mixtures with 125 pg of male DNA in the presence of 50 ng of female DNA. The ability to tolerate polymerase chain reaction inhibitors commonly contained in forensic casework samples demonstrated the applicability and robustness of the Y34plex. Compared with the Yfiler Plus kit, the novel panel showed an increased power of discrimination in Chinese Wuxi Han population (n = 434). The overall haplotype diversity of the Y34plex was 0.999606, whereas DC value was 0.956221, which is suitable for use on forensic paternal investigation.

Molecular characterization of 23 Y chromosomal STR markers in the Gurjar population of National Capital Region (NCR), India.

In the present study, DNA samples of 202 unrelated male individuals of Gurjar population were evaluated for the molecular diversity at 23 Y chromosomal Y-STR markers. Out of selected individuals, results showed 143 unique haplotypes. Highest degree of gene diversity (GD), polymorphic information content (PIC), and power of discrimination (PD) was observed as 0.7941, 0.7590, and 0.7902, respectively, for the locus DYS385a/b. Haplotype diversity (HD), gene diversity (GD), polymorphic information content (PIC), and power of discrimination (PD) was found to be 0.7079, 0.999999999989, 0.9999999996, and 0.999999999986, respectively, for the studied 23 Y-STR markers. Allele 11 of locus DYS392 was found to be the most frequent allele with the frequency of 0.762. In inter-population relationship, studied population showed genetic relatedness with the population of Jammu and Kashmir, India, and Ladakh, India. The haplotype data of the present study will not only enrich the existing Indian Y-STR data but will also be useful for forensic DNA application.

Retrofitting massively parallel sequencing (MPS) for HLA-DQA1 and polymarker (PM) in forensic casework.

Genotype profiling has played a major role in forensics for decades. The technology for detection and discrimination has advanced substantially, from serology to DNA sequence analysis. Currently, there may be situations where there is a need for re-analysis of forensic DNA data that was produced using methodology that is no longer available. An example of this is the allele-specific oligonucleotide hybridization assays used in the 1990s. In the study presented herein, we have developed a multiplex system combining PCR and massively parallel sequencing (MPS) technologies to identify DNA polymorphisms. Our results are consistent with those found in the widely utilized AmpliType PM + DQA1 Amplification and Typing Kit originally marketed by Perkin Elmer. During the course of our studies, it became apparent that paralogous genes for two of the loci, GYPA and HBG2 (formerly HBGG), could have confounded the interpretation of the original assays, and we describe the technical solutions we developed to overcome ambiguity in genotype assignment. This study results in a novel resource enabling the re-analysis of DNA profiling results produced decades past using current day technology.

CRISPR-CasB technology in forensic DNA analysis: challenges and solutions.

CRISPR-Cas technology has revolutionized the field of biotechnology with its precise therapeutic use from genetic as well as infectious diseases point of view. This technology is rapidly evolving to single tool enabling site-directed cut in the genome and highly specific activation or inhibition of gene expression or the exchange of single bases. Besides clinical applications, CRISPR-Cas technology has also shown promising use in the field of forensic DNA analysis. Enrichment of targeted genetic marker for identification followed by sequencing and non-PCR-dependent technique ensures the use of CRISPR-Cas technology in challenging forensic biological samples. The use of this advanced technology is also deemed helpful in mixed profile attribution, mostly in LCN contributors and the generation of a useful DNA profile in degraded samples. Besides its useful applications in forensic DNA analysis, CRISPR-Cas technology poses a huge threat from the generation of ghost DNA profiles by modification/alteration of target genetic markers. Forensic DNA analysts should carry out analysis of additional markers such as non-CODIS markers, Y-, X-chromosome markers, and mitochondrial DNA sequencing in a suspected ghost DNA profile case. KEY POINTS: • CRISPR-Cas9 technique is useful in analyzing LCN, mixed and degraded samples • Alteration of DNA using this technique can lead to generation of ghost DNA profiles • Alternative genetic markers and methylation pattern may detect a ghost DNA profile.

Forensic features and phylogenetic analyses of the population of Nayagarh (Odisha), India using 23 Y-STRs.

AIM: The present study was designed to explore the STR diversity and genomic history of the inhabitants of Nayagarh district of Odisha, India. We also tested the proficiency of the most recent, new generation PowerPlexR Y23 multiplex system for forensic characterisation and to decipher the phylogenetic affinities. SUBJECTS AND METHODS: The genetic diversity and polymorphism among 236 healthy unrelated male volunteers from Nayagarh district of Odisha, India was investigated. This investigation was carried out via 23 Y-chromosomal STRs using capillary electrophoresis. RESULT: A total 223 unique haplotypes were reported. Discrimination capacity (DC), gene diversity (GD) and power of discrimination (PD) were observed as 0.945, 0.999999999998333, and 0.99999999999794, respectively. Polymorphic information content (PIC) and matching probability (PM) were reported as 0.999999999925535 and 2.06 × 10-12, respectively. Simultaneously, the haplogroup analysis characterised with C2, E1b1a, E1b1b, G2a, H1, I2a, J2a, J2b, L, O, O1, O2, Q, R1a, R2, and T haplogroups, disclosing the possible geographical relatedness of the studied population to different areas of the world. CONCLUSION: Phylogenetic analysis with previously reported Indian and Asian populations showed the genetic closeness of the studied population to different Indian populations and the Bangladeshi population of Dhaka, whereas the Bhotra population of Odisha and Han population of China showed much less genetic affinity.

Accumulation of endogenous and exogenous nucleic acids in "Touch DNA" components on hands.

Successful forensic DNA profiling from handled items is increasingly routine in casework. This "touch DNA" is thought to contain both cellular and acellular nucleic acid sources. However, there is little clarity on the origins or characteristics of this material. The cellular component consists of anucleate, terminally differentiated corneocytes (assumed to lack DNA), and the occasional nucleated cell. The acellular DNA source is fragmentary, presumably cell breakdown products. This study examines the relative contributions each component makes to the hand-secretions (endogenous) and hand-accumulations (exogenous) by recovering rinses from the inside and outside of worn gloves. Additionally, cellular and acellular DNA was measured at timepoints up to 2 h after hand washing, both with and without interim contact. Microscopic examination confirmed cell morphology and presence of nucleic acids. Following the novel application of a hair keratinocyte lysis method and plasma-DNA fragment purification to hand rinse samples, DNA profiles were generated from both fractions. Exogenous cell-free DNA is shown to be a significant source of touch DNA, which reaccumulates quickly, although its amplifiable nuclear alleles are limited. Endogenous DNA is mostly cellular in origin and provides more allelic information consistently over time.

Match statistics for sequence-based alleles in profiles from forensic PCR-mps kits.

The first autosomal sequence-based allele (aka SNP-STR haplotype) frequency database for forensic massively parallel sequencing (MPS) has been published, thereby removing one of the remaining barriers to implementing MPS in casework. The database was developed using a specific set of flank trim sites. If different trim sites or different kits with different primers are used for casework, then SNP-STR haplotypes may be detected that do not have frequencies in the database. We describe a procedure to address calculation of match probabilities when casework samples are generated using an MPS kit with different trim sites than those present in the relevant population frequency database. The procedure provides a framework for comparison of any MPS kit or database combination while also accommodating comparison of MPS and CE profiles.

Towards developing forensically relevant single-cell pipelines by incorporating direct-to-PCR extraction: compatibility, signal quality, and allele detection.

Current analysis of forensic DNA stains relies on the probabilistic interpretation of bulk-processed samples that represent mixed profiles consisting of an unknown number of potentially partial representations of each contributor. Single-cell methods, in contrast, offer a solution to the forensic DNA mixture problem by incorporating a step that separates cells before extraction. A forensically relevant single-cell pipeline relies on efficient direct-to-PCR extractions that are compatible with standard downstream forensic reagents. Here we demonstrate the feasibility of implementing single-cell pipelines into the forensic process by exploring four metrics of electropherogram (EPG) signal quality-i.e., allele detection rates, peak heights, peak height ratios, and peak height balance across low- to high-molecular-weight short tandem repeat (STR) markers-obtained with four direct-to-PCR extraction treatments and a common post-PCR laboratory procedure. Each treatment was used to extract DNA from 102 single buccal cells, whereupon the amplification reagents were immediately added to the tube and the DNA was amplified/injected using post-PCR conditions known to elicit a limit of detection (LoD) of one DNA molecule. The results show that most cells, regardless of extraction treatment, rendered EPGs with at least a 50% true positive allele detection rate and that allele drop-out was not cell independent. Statistical tests demonstrated that extraction treatments significantly impacted all metrics of EPG quality, where the Arcturus® PicoPure™ extraction method resulted in the lowest median allele drop-out rate, highest median average peak height, highest median average peak height ratio, and least negative median values of EPG sloping for GlobalFiler™ STR loci amplified at half volume. We, therefore, conclude the feasibility of implementing single-cell pipelines for casework purposes and demonstrate that inferential systems assuming cell independence will not be appropriate in the probabilistic interpretation of a collection of single-cell EPGs.

Progress in the implementation of massively parallel sequencing for forensic genetics: results of a European-wide survey among professional users.

A European-wide online survey was conducted to generate an overview on the state-of-the-art using massively parallel sequencing (MPS) platforms for forensic DNA analysis and DNA phenotyping among forensic practitioners in Europe. The survey was part of the dissemination activities of the "VISible Attributes through GEnomics - VISAGE" Horizon 2020 funded European research project [30], in preparation of a series of educational training activities. A total of 105 replies from 32 European countries representing participants from police, governmental, academic, and private laboratories providing professional services in the field of forensic genetics were included in the final analysis. Of these, 73% already own an MPS platform or are planning to acquire one within the next 1-2 years. One-third of the participants have already carried out MPS-based STR sequencing, identity, or ancestry SNP typing. A total of 23-40% of participants are planning to explore all FDP applications showing the overall very high interest in using MPS for the whole range of forensic MPS markers and applications. About 50% of the participants have previously gathered experience using forensic DNA phenotyping (FDP) markers based on conventional (i.e., not MPS-based) DNA typing methods. A total of 55% of the participants have attended training on the general use of MPS technology, but 36% have received no training whatsoever. Accordingly, 90% have expressed high or medium interest to attend training on the analysis and interpretation of DNA phenotyping data for predicting appearance, ancestry, and age. The results of our survey will provide valuable information for organizing relevant training workshops on all aspects of MPS-based DNA phenotyping for the forensic genetics scientific community.

Population genetic data for 21 autosomal STR loci in the Azerbaijani population using the Globalfiler™ kit.

The Republic of Azerbaijan is located in the southern Caucasus mountains, a region which is linguistically and ethnically diverse. We report allele frequency data for 21 autosomal loci from the Globalfiler™ kit in the Azerbaijani population using 467 individuals from Baku. Exact tests for Hardy Weinberg Equilibrium and Linkage Equilibrium were conducted, and all forensic parameters were estimated. High levels of Expected Heterozygosity HE were seen, with a minimum of 0.637 for TPOX and a maximum of 0.949 for SE33. Polymorphism Information Content (PIC) values for all STR loci were high, ranging from 0.587 for TPOX to 0.947 for SE33. Matching probability (MP) estimates ranged from 0.006 for SE33 to 0.178 for TPOX. Power of Discrimination (PD) values for most of the tested loci (17/21) were ≥ 0.9. The Combined Matching Probability (CMP) and the Combined Power of Discrimination (CPD) for all 21 loci were 7.84 × 10-27 and 1.0 respectively. Exact tests for population differentiation using all available Globalfiler™ datasets from across Europe and Asia reveal a general trend of higher differentiation with increasing geographical separation, but there is a need for additional population data from neighboring regions of Azerbaijan.

Prediction of eye and hair pigmentation phenotypes using the HIrisPlex system in a Brazilian admixed population sample.

Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual's ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution.