Maria João Porto

A GEP-ISFG collaborative study on the optimization of an X-STR decaplex: data on 15 Iberian and Latin American populations

In a collaborative work carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG), a polymerase chain reaction multiplex was optimized in order to type ten X-chromosome short tandem repeats (STRs) in a single reaction, including: DXS8378, DXS9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7133, GATA172D05, GATA31E08, and DXS7423. Using this X-decaplex, each 17 of the participating laboratories typed a population sample of approximately 200 unrelated individuals (100 males and 100 females). In this work, we report the allele frequencies for the ten X-STRs in 15 samples from Argentina (Buenos Aires, Córdoba, Río Negro, Entre Ríos, and Misiones), Brazil (São Paulo, Rio de Janeiro, Paraná, and Mato Grosso do Sul), Colombia (Antioquia), Costa Rica, Portugal (Northern and Central regions), and Spain (Galicia and Cantabria). Gene diversities were calculated for the ten markers in each population and all values were above 56%. The average diversity per locus varied between 66%, for DXS7133, and 82%, for DXS6809. For this set of STRs, a high discrimination power was obtained in all populations, both in males (≥1 in 5u2009×u2009105) and females (≥1 in 3u2009×u2009109), as well as high mean exclusion chance in father/daughter duos (≥99.953%) and in father/mother/daughter trios (≥99.999%). Genetic distance analysis showed no significant differences between northern and central Portugal or between the two Spanish samples from Galicia and Cantabria. Inside Brazil, significant differences were found between Rio de Janeiro and the other three populations, as well as between São Paulo and Paraná. For the five Argentinean samples, significant distances were only observed when comparing Misiones with Entre Ríos and with Río Negro, the only two samples that do not differ significantly from Costa Rica. Antioquia differed from all other samples, except the one from Río Negro.

Collaborative EDNAP exercise on the IrisPlex system for DNA-based prediction of human eye colour

The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on FTA cards by the organising laboratory together with eye colour phenotypes; 99.4% of the genotypes were correctly reported and 99% of the eye colour phenotypes were correctly predicted. Task 2 involved the assessment of 5 DNA samples extracted by the host laboratory from simulated casework samples, artificially degraded, and provided to the participants in varying DNA concentrations. For this task, 98.7% of the genotypes were correctly determined and 96.2% of eye colour phenotypes were correctly inferred. For Tasks 1 and 2 together, 99.2% (1875) of the 1890 genotypes were correctly generated and of the 15 (0.8%) incorrect genotype calls, only 2 (0.1%) resulted in incorrect eye colour phenotypes. The voluntary Task 3 involved participants choosing their own test subjects for IrisPlex genotyping and eye colour phenotype inference, while eye photographs were provided to the organising laboratory and judged; 96% of the eye colour phenotypes were inferred correctly across 100 samples and 19 laboratories. The high success rates in genotyping and eye colour phenotyping clearly demonstrate the reproducibility and the robustness of the IrisPlex assay as well as the accuracy of the IrisPlex model to predict blue and brown eye colour from DNA. Additionally, this study demonstrates the ease with which the IrisPlex system is implementable and applicable across forensic laboratories around the world with varying pre-existing experiences.

Genetic portrait of Lisboa immigrant population from Angola with mitochondrial DNA

Portugal has been considered a country of emigrants, nevertheless in the past decades the number of immigrants has grown throughout all the country. This migratory flux has contributed to a raise of heterogeneity at multiple levels. According to statistical data, at the end of 2012 the total number of Angolan immigrants in Portugal equalled about 20,000 individuals. A territorial predominance has been found for the metropolitan region of Lisboa. Angola is a country located in the Atlantic coast of Africa. The presence of Bantu people and the colonisation by Portuguese people on Angolan territory are considered to be the major modulators of the genetic patterns in Angola. Mitochondrial DNA is known for its features that enable an approach to the study of human origin and evolution, as well to the different migration pathways of populations. This genetic marker can also contribute to ascertaining the identity of individuals in forensic cases. The main aim of this study was to determine the genetic structure of the Angolan immigrant population living in Lisboa. Therefore, a total of 173 individuals, inhabitants in Lisboa, nonrelated and with Angolan ancestry were studied. Total control region of mitochondrial DNA was amplified from position 16,024 to position 576 using two pairs of primers - L15997/H016 and L16555/H639. The majority of the identified haplotypes belong to mtDNA lineages known to be specific of the sub-Saharan region. Our results show that this immigrant population inhabitant in Lisboa presents a genetic profile that is characteristic of African populations. This study also demonstrates the genetic diversity that this immigrant population introduces in Lisboa. This does not contradict the historical data concerning colonization of Angola, since this was made mainly by male European individuals, who did not contribute with their maternal information of mtDNA. Lisboa immigrant population from Angola can be accessed via EMPOP dataset with accession number EMPOP662.

GHEP-ISFG collaborative simulated exercise for DVI/MPI: Lessons learned about large-scale profile database comparisons

The GHEP-ISFG Working Group has recognized the importance of assisting DNA laboratories to gain expertise in handling DVI or missing persons identification (MPI) projects which involve the need for large-scale genetic profile comparisons. Eleven laboratories participated in a DNA matching exercise to identify victims from a hypothetical conflict with 193 missing persons. The post mortem database was comprised of 87 skeletal remain profiles from a secondary mass grave displaying a minimal number of 58 individuals with evidence of commingling. The reference database was represented by 286 family reference profiles with diverse pedigrees. The goal of the exercise was to correctly discover re-associations and family matches. The results of direct matching for commingled remains re-associations were correct and fully concordant among all laboratories. However, the kinship analysis for missing persons identifications showed variable results among the participants. There was a group of laboratories with correct, concordant results but nearly half of the others showed discrepant results exhibiting likelihood ratio differences of several degrees of magnitude in some cases. Three main errors were detected: (a) some laboratories did not use the complete reference family genetic data to report the match with the remains, (b) the identity and/or non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, and (c) many laboratories did not properly evaluate the prior odds for the event. The results suggest that large-scale profile comparisons for DVI or MPI is a challenge for forensic genetics laboratories and the statistical treatment of DNA matching and the Bayesian framework should be better standardized among laboratories.

GHEP-ISFG collaborative exercise on mixture profiles (GHEP-MIX06). Reporting conclusions: Results and evaluation

One of the main goals of the Spanish and Portuguese-Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. Due to this fact, GHEP-ISFG holds different working commissions that are set up to develop activities in scientific aspects of general interest. One of them, the Mixture Commission of GHEP-ISFG, has organized annually, since 2009, a collaborative exercise on analysis and interpretation of autosomal short tandem repeat (STR) mixture profiles. Until now, six exercises have been organized. At the present edition (GHEP-MIX06), with 25 participant laboratories, the exercise main aim was to assess mixture profiles results by issuing a report, from the proposal of a complex mock case. One of the conclusions obtained from this exercise is the increasing tendency of participating laboratories to validate DNA mixture profiles analysis following international recommendations. However, the results have shown some differences among them regarding the edition and also the interpretation of mixture profiles. Besides, although the last revision of ISO/IEC 17025:2017 gives indications of how results should be reported, not all laboratories strictly follow their recommendations. Regarding the statistical aspect, all those laboratories that have performed statistical evaluation of the data have employed the likelihood ratio (LR) as a parameter to evaluate the statistical compatibility. However, LR values obtained show a wide range of variation. This fact could not be attributed to the software employed, since the vast majority of laboratories that performed LR calculation employed the same software (LRmixStudio). Thus, the final allelic composition of the edited mixture profile and the parameters employed in the software could explain this data dispersion. This highlights the need, for each laboratory, to define through internal validations its criteria for editing and interpreting mixtures, and to continuous train in software handling.

Study of InDel genetic markers with forensic and ancestry informative interest in PALOP's immigrant populations in Lisboa.

The migratory phenomenon in Portugal has become one of the main factors for the genetic variability. In the last few years, a new class of autosomal insertion/deletion markers—InDel—has attracted interest in forensic genetics. Since there is no data for InDel markers of Portuguese-speaking African countries (PALOP) immigrants living in Lisboa, our aim is the characterization of those groups of individuals by typing them with at least 30 InDel markers and to compare different groups of individuals/populations. We studied 454 bloodstain samples belonging to immigrant individuals from Angola, Guinea-Bissau, and Mozambique. DNA extraction was performed with the Chelex® 100 method. After extraction, all samples were typed with the Investigator® DIPplex method. Through the obtained results, allelic frequencies show that all markers are at Hardy-Weinberg equilibrium, and we can confirm that those populations show significant genetic distances between themselves, between them, and the host Lisboa population. Because of this, they introduce genetic variability in Lisboa population.

Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.

Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.

A65 Mitochondrial DNA studies of Lisbon immigrants from Portuguese speaking African countries

The use of next-generation sequencing for discovery of viruses has yielded vast amounts of known and putative viral reads. The computational analysis of the reads, however, is quite a challenge, in particular the classification of reads to viral taxa. This is illustrated by the many computational tools that have been devised and new tools that appear monthly. These provide opportunities for other researchers, but the large numbers make it hard for virologists to pick a tool that suits their own study. To facilitate this choice and guide users to through the forest of computational pipelines, we have surveyed publications describing fifty tools, inventoried their approaches and scored their methods, user-friendliness, validation, and other performance criteria for diagnostics, outbreak source tracing, virus discovery, and virome profiling. The tools have variable approaches to the classification of viral reads, but they all rely on searching (i.e., homology (44/50 pipelines) and composition search (8/50)) through reference databases (e.g., nucleotide databases, protein databases, or virus-specific databases). Some pipelines include quality control/pre-processing of reads (23/ 50), filtering non-viral reads (20/50), and de novo assembly (18/ 50) before the search, and checking and correcting classifications with phylogenetic or statistical methods after the search step (8/50). Furthermore, some are tailor-made for particular studies; others are more generally applicable. Few tools provide a graphical user interface, and when they do they are often online, which increases the ease of use. Reported runtimes vary greatly—from several minutes per sample to days; newer tools are often faster than old ones. Moreover, some tools have been validated in wet-lab experiments or compared to other tools with in silico benchmark tests. The overview of pipelines is presented on the COMPARE website (https://compare.cbs.dtu.dk/in ventory#pipeline). A decision tree is provided separately to help virologists with any level of bioinformatics expertise select suitable analysis tools. The next step will be to benchmark the most promising tools with the COMPARE and VIROGENESIS projects to better assess their performance for diagnostics and surveillance studies.

PowerPlex® fusion 6C system: internal validation study

ABSTRACT This work is aimed at describing the proceedings and parameters used to validate PowerPlex® Fusion 6C System, the polymerase chain reaction (PCR) amplification kit by Promega, for posterior implementation in the laboratorial routine of the Forensic Genetic Service. The PowerPlex® Fusion 6C System allows multiplex PCR, through simultaneous amplification and posterior detection by fluorescence of 27 loci. Characterization of the kit was made according to the laboratorys internal validation procedure based on validation guidelines from Scientific Working Group on DNA Analysis Methods. Some parameters were evaluated, such as specificity, analytical thresholds, sensitivity, precision, mixture studies, DNA control samples, a proficiency test and changes in the PCR-based procedures: final reaction volume and cycle number, changes in the reaction mixture for direct amplification. This kit proved to be very robust and the results are in concordance with previous developmental validation by the manufacturer. In some parameters, the results were better than expected.