Instituto de Ciencias Forenses (INCIFOR)

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Relationship Between Vertigo and Consumption of Psychotropic Drugs: A Prospective Case–Control Study
(MDPI, 2025-04-08) Sánchez Sellero, Inés; Soto Varela, Andrés; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Departamento de Cirurxía e Especialidades Médico-Cirúrxicas
Background/Objectives: The association between vestibular symptoms and psychological distress has been previously studied, mainly with the use of questionnaires. The purpose of this study is to compare the consumption of psychotropic drugs between a group of patients with vertigo and a control group. Methods: A prospective cross-sectional, observational, case–control study was carried out, including 506 patients (232 with Ménière’s disease, 79 with vestibular migraine, 34 with vestibular neuritis, and 161 with benign paroxysmal positional vertigo). In total, 253 participants were included in the control group. Both groups were comparable regarding age, sex, and history of previous psychiatric diseases. Results: The percentage of patients with vertigo who consumed psychotropic drugs (41.3%) was higher than the percentage of the control group who did so (26.9%) (Fisher’s exact test, p < 0.0001; OR = 1.914, CI95% (1.377; 2.662)). The mean number of psychotropic drugs consumed was also higher (Mann–Whitney test, p = 0.0003) in cases (0.68 ± 0.959) than in controls (0.47 ± 0.889). This higher consumption in the group of patients with vertigo was found for all pharmacological groups studied, being especially relevant regarding “anxiolytics and hypnotics and sedatives” and “antidepressants”. No statistically significant differences in the consumption of psychotropic drugs between types of vestibular disorders were observed. The longer the symptoms were present, the higher the prevalence of psychotropic drug use was observed. Conclusions: A relationship between vertigo and consumption of psychotropic drugs was found. Recording the consumption of these drugs is proposed as an objective method to better understand the psychological distress that patients with vertigo may suffer from.
Evaluating the trends and impact of COVID-19 on illicit drug and benzodiazepine use in drivers: A retrospective large-scale study based on oral fluid testing
(Elsevier, 2026-02-26) Blanco Ces, Miriam; Lendoiro Belío, Elena; Cruz Landeira, Angelines; Cobo Golpe, María; López Rabuñal, Ángela; Castro Ríos, Ana de; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Background: The COVID-19 pandemic disrupted global drug markets, but consumption soon returned to pre- pandemic levels. Continuous monitoring of drug use trends is essential for effective public health responses. Methods: A total of 29,397 oral fluid specimens from roadside drug tests across Spain (January 2019–July 2024) were sent to the Toxicology Laboratory of the Institute of Forensic Sciences, University of Santiago de Compostela, for LC-MS/MS confirmation of on-site positives. Results were stratified into five periods to assess drug use trends and the impact of COVID-19. Results: Over 90% of drivers were male, and 85% were under 45 years old. Overall, 69.9% of samples were positive for cannabis, 64.9% for cocaine, 13.7% for amphetamines, 10.6% for opiates, 6.5% for ketamine, 5.4% for methadone and 6.6% for benzodiazepines/zolpidem; 56.3% showed poly-drug use. Cannabis use was higher in men, while amphetamines and benzodiazepines were more frequent in women (p < 0.001). Due to regional variability in drug use patterns and sample distribution, trends were analyzed across five geographic regions. Globally, during the strict lockdown, cocaine, opiates, methadone and benzodiazepines peaked, while cannabis and amphetamines declined, and ketamine remained stable. In the final period, cannabis reached its highest levels, and ketamine showed a marked increase. Cocaine and amphetamines returned to pre-COVID levels, while opiates, methadone and benzodiazepines declined. Statistically significant differences across the studied periods were observed in the different regions. Specifically, in the Northwest for opiates, methadone, and benzodiazepines (p < 0.001), as well as for cannabis (p < 0.05); in the East for opiates, amphetamines, cocaine, and ketamine (p < 0.001); in the Center for cocaine (p < 0.001) and ketamine (p < 0.05); and in the South and Islands for cocaine (p < 0.05). Conclusions: Although a slight impact on drug use was observed during the strict lockdown, consumption increased again for all substances (particularly ketamine), except for opiates, methadone and benzodiazepines.
Possible adverse effects of mining activity on the neurocognitive development of children in the area of Cerro de Pasco (Perú)
(Elsevier, 2026-05) Carreiro-DaCunha, Elton; Ordóñez Mayán, Lucía; Bianchini, Flaviano; Muñoz Barús, José Ignacio; Universidade de Santiago de Compostela. Departamento de Psiquiatría, Radioloxía, Saúde Pública, Enfermaría e Medicina; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Over the last 10 years there have been a number of studies examining the effects of exposure to environmental metal pollution on the population of the area of Cerro de Pasco (Peru). These have documented the prolonged pollution of the area caused by mining activity and recorded its pathological effects on the exposed population. The present work reports associations between the concentrations of metals in the hair of the area's children and their cognitive development, investigates the neurocognitive effects of exposure, and examines the change in environmental metal concentrations over time. Significant differences in hair metal concentrations were detected between exposed (case) and non-exposed (control) populations; in the former, the mean arsenic concentration was three times that of the latter, the cadmium concentration was double, and that of lead six times that of the latter. The mean total IQ of the exposed children was 12.3 points lower than those who were not exposed. Significant correlations were detected between the lead, cadmium, arsenic, manganese and antimony concentrations of the children's (combined exposed and non-exposed) hair and TIQ. In the exposed population, marked increases in hair metal concentrations were recorded between 2016 and 2018 (200 %), later falling by 2021 (though still exceeding the 2016 concentrations). Multivariate analyses involving big data are required to determine the covariables that influence the development of TIQ in exposed children, and to determine whether high toxic metal concentrations are an independent risk factor for cognitive deficit.
Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers
(Multidisciplinary Digital Publishing Institute (MDPI), 2025-01-01) Martelo Vidal, Laura; Vázquez Mera, Sara; Miguéns Suárez, Pablo; Domínguez Arca, Vicente; Gómez Carballa, Alberto; Salas Ellacuriaga, Antonio; González Barcala, Francisco Javier; Salgado Castro, Francisco Javier; Nieto Fontarigo, Juan José; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Departamento de Psiquiatría, Radioloxía, Saúde Pública, Enfermaría e Medicina
This study aims to develop a protocol for respiratory disease-associated biomarker discovery by combining urine proteome studies with urinary exosome components analysis (i.e., miRNAs). To achieve this, urine was DTT treated to decrease uromodulin, then concentrated and ultracentrifuged. Proteomic analyses of exosome-free urine were performed using LC-MS/MS. Simultaneously, miRNA expression from urine exosomes was measured using either RTqPCR (pre-amplification) or nCounter Nanostring (non-amplication) analyses. We detected 548 different proteins in exosome-free urine samples (N = 5) with high confidence (FDR < 1%), many of them being expressed in different non-renal tissues. Specifically, lung-related proteins were overrepresented (Fold enrichment = 1.31; FDR = 0.0335) compared to whole human proteome, and 10–15% were already described as protein biomarkers for several pulmonary diseases. Urine proteins identified belong to several functional categories important in respiratory pathology. We could confirm the expression of miRNAs previously connected to respiratory diseases (i.e., miR-16-5p, miR-21-5p, miR-146a-5p, and miR-215-5p) in urine exosomes by RTqPCR. Finally, we detected 333 miRNAs using Nanostring, 15 of them up-regulated in T2high asthma (N = 4) compared to T2low asthma (N = 4) and healthy subjects (N = 4). Therefore, this protocol combining the urinary proteome (exosome free) with the study of urinary exosome components (i.e., miRNAs) holds great potential for molecular biomarker discovery of non-renal and particularly respiratory pathologies.
Forensic SNP Genotyping with SNaPshot: Technical Considerations for the Development and Optimization of Multiplexed SNP Assays
(Forensic Science Review, 2017-01-01) Fondevila, M.; Børsting, C.; Phillips, C; Puente Vila, María del Carmen de la; Santos, C.; EUROFORGEN-NoE Consortium; Carracedo Álvarez, Ángel; Morling, N.; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
This review explores the key factors that influence the optimization, routine use, and profile interpretation of the SNaPshot single-base extension (SBE) system applied to forensic single-nucleotide polymorphism (SNP) genotyping. Despite being a mainly complimentary DNA genotyping technique to routine STR profiling, use of SNaPshot is an important part of the development of SNP sets for a wide range of forensic applications with these markers, from genotyping highly degraded DNA with very short amplicons to the introduction of SNPs to ascertain the ancestry and physical characteristics of an unidentified contact trace donor. However, this technology, as resourceful as it is, displays several features that depart from the usual STR genotyping far enough to demand a certain degree of expertise from the forensic analyst before tackling the complex casework on which SNaPshot application provides an advantage. In order to provide the basis for developing such expertise, we cover in this paper the most challenging aspects of the SNaPshot technology, focusing on the steps taken to design primer sets, optimize the PCR and single-base extension chemistries, and the important features of the peak patterns observed in typical forensic SNP profiles using SNaPshot. With that purpose in mind, we provide guidelines and troubleshooting for multiplex-SNaPshot-oriented primer design and the resulting capillary electrophoresis (CE) profile interpretation (covering the most commonly observed artifacts and expected departures from the ideal conditions).
Development of the VISAGE enhanced tool and statistical models for epigenetic age estimation in blood, buccal cells and bones
(Aging, 2021-03-11) Woznial, Anna; Heidegger, Antonia; Piniewska-Róg, Danuta; Pośpiech, Ewelina; Xavier, Catarina; Pisarek, Aleksandra; Kartasińska, Ewa; Boroń, Michal; Freire Aradas, Ana María; Wojtas, Marta; Puente Vila, María del Carmen de la; Niederstätter, Harald; Płoski, Rafal; Spólnicka, Magdalena; Kayser, Manfred; Phillips, Christopher P.; Parson, Walther; Branicki, Wojciech; VISAGE Consortium; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
DNA methylation is known as a biomarker for age with applications in forensics. Here we describe the VISAGE (VISible Attributes through GEnomics) Consortium’s enhanced tool for epigenetic age estimation in somatic tissues. The tool is based on eight DNA methylation markers (44 CpGs), bisulfite multiplex PCR followed by sequencing on the MiSeq FGx platform, and three statistical prediction models for blood, buccal cells and bones. The model for blood is based on six CpGs from ELOVL2, MIR29B2CHG, KLF14, FHL2, TRIM59 and PDE4C, and predicts age with a mean absolute error (MAE) of 3.2 years, while the model for buccal cells includes five CpGs from PDE4C, MIR29B2CHG, ELOVL2, KLF14 and EDARADD and predicts age with MAE of 3.7 years, and the model for bones has six CpGs from ELOVL2, KLF14, PDE4C and ASPA and predicts age with MAE of 3.4 years. The VISAGE enhanced tool for age estimation in somatic tissues enables reliable collection of DNA methylation data from small amounts of DNA using a sensitive multiplex MPS assay that provides accurate estimation of age in blood, buccal swabs, and bones using the statistical model tailored to each tissue
Development and Evaluation of the Ancestry Informative Marker Panel of the VISAGE Basic Tool
(MDPI, 2021-08-22) Puente Vila, María del Carmen de la; Ruiz Ramírez, Jorge; Ambroa Conde, Adrián; Álvarez Dios, José Antonio; Freire Aradas, Ana María; Mosquera Miguel, Ana; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Phillips, Christopher; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
We detail the development of the ancestry informative single nucleotide polymorphisms (SNPs) panel forming part of the VISAGE Basic Tool (BT), which combines 41 appearance predictive SNPs and 112 ancestry predictive SNPs (three SNPs shared between sets) in one massively parallel sequencing (MPS) multiplex, whereas blood-based age analysis using methylation markers is run in a parallel MPS analysis pipeline. The selection of SNPs for the BT ancestry panel focused on established forensic markers that already have a proven track record of good sequencing performance in MPS, and the overall SNP multiplex scale closely matched that of existing forensic MPS assays. SNPs were chosen to differentiate individuals from the five main continental population groups of Africa, Europe, East Asia, America, and Oceania, extended to include differentiation of individuals from South Asia. From analysis of 1000 Genomes and HGDP-CEPH samples from these six population groups, the BT ancestry panel was shown to have no classification error using the Bayes likelihood calculators of the Snipper online analysis portal. The differentiation power of the component ancestry SNPs of BT was balanced as far as possible to avoid bias in the estimation of co-ancestry proportions in individuals with admixed backgrounds. The balancing process led to very similar cumulative population-specific divergence values for Africa, Europe, America, and Oceania, with East Asia being slightly below average, and South Asia an outlier from the other groups. Comparisons were made of the African, European, and Native American estimated co-ancestry proportions in the six admixed 1000 Genomes populations, using the BT ancestry panel SNPs and 572,000 Affymetrix Human Origins array SNPs. Very similar co-ancestry proportions were observed down to a minimum value of 10%, below which, low-level co-ancestry was not always reliably detected by BT SNPs. The Snipper analysis portal provides a comprehensive population dataset for the BT ancestry panel SNPs, comprising a 520-sample standardised reference dataset; 3445 additional samples from 1000 Genomes, HGDP-CEPH, Simons Foundation and Estonian Biocentre genome diversity projects; and 167 samples of six populations from in-house genotyping of individuals from Middle East, North and East African regions complementing those of the sampling regimes of the other diversity projects.
Evaluation of the Qiagen 140-SNP forensic identification multiplex for massively parallel sequencing
(Elsevier, 2017-01-27) Puente Vila, María del Carmen de la; Phillips, Christopher P.; Santos, C.; Fondevila, Manuel; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
A new forensic 140-SNP genotyping system from Qiagen, designed for massively parallel sequencing (MPS) analysis, was evaluated using the Ion PGM™ MPS system. Assessments consisted of the sequencing of: established control DNAs that had been previously genotyped with alternative PCR and library preparation kits supplied by Thermo Fisher Scientific for the Ion PGM™ system; a simple set of artificial DNA mixtures; DNA extracted from a degraded femur; and a dilution series to gauge forensic sensitivity. In addition to the reagents for the DNA target capture PCR and library preparation, Qiagen offer an alternative sequence analysis software system (Workbench), which was assessed alongside the Ion PGM™ Genotyper software for forensic MPS analysis. The Qiagen SNP genotyping system produced full genotyping concordance with previous data obtained with a similar SNP panel on the Ion PGM™ and in comparison to genotypes listed for 139 of the 140 SNPs in 1000 Genomes. The workbench software was as reliable as Genotyper in calling genotypes, although scrutiny of sequence data with IGV revealed the problem of sequence misalignment plagues a small proportion of the 140 SNPs in the Qiagen panel, a problem already recognized in multiple MPS studies of the same markers in alternative kits. The potential for genotype miscalls from sequence misalignment in certain SNPs will require manual inspection in cases where low-level or degraded DNA reduces the sequence coverage to a point where misalignment influences individual SNP genotype quality
The global AIMs nano set: A 31-plex SNaPshot assay of ancestry-informative SNPs
(Elsevier, 2016-01-25) Puente Vila, María del Carmen de la; Santos, C.; Fondevila, Manuel; Manzo, L; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Phillips, Christopher P.; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
A 31-plex SNaPshot assay, named 'Global AIMs Nano', has been developed by reassembling the most differentiated markers of the EUROFORGEN Global AIM-SNP set. The SNPs include three tri-allelic loci and were selected with the goal of maintaining a balanced differentiation of: Africans, Europeans, East Asians, Oceanians and Native Americans. The Global AIMs Nano SNP set provides higher divergence between each of the five continental population groups than previous small-scale AIM sets developed for forensic ancestry analysis with SNaPshot. Both of these characteristics minimise potential bias when estimating co-ancestry proportions in individuals with admixed ancestry; more likely to be observed when using markers disproportionately informative for only certain population group comparisons. The optimised multiplex is designed to be easily implemented using standard capillary electrophoresis regimes and has been used to successfully genotype challenging forensic samples from highly degraded material with low level DNA. The ancestry predictive performance of the Global AIMs Nano set has been evaluated by the analysis of samples previously characterised with larger AIM sets
Inter-laboratory evaluation of the EUROFORGEN global ancestry-informative SNP panel by massively parallel sequencing using the Ion PGM™
(Elsevier, 2016-04-21) Eduardoff, M.; Gross, T. E.; Santos, C.; Puente Vila, María del Carmen de la; Ballard, David J.; Strobl, C.; Børsting, Claus; Morling, Niels; Fusco, L.; Hussing, C.; Egyed, B.; Souto, L.; Uacyisrael, J.; Syndercombe Court, Y. Denise; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Schneider, Peter M.; Parson, Walther; Phillips, Christopher P.; EUROFORGEN-NoE Consortium; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
The EUROFORGEN Global ancestry-informative SNP (AIM-SNPs) panel is a forensic multiplex of 128 markers designed to differentiate an individual's ancestry from amongst the five continental population groups of Africa, Europe, East Asia, Native America, and Oceania. A custom multiplex of AmpliSeq™ PCR primers was designed for the Global AIM-SNPs to perform massively parallel sequencing using the Ion PGM™ system. This study assessed individual SNP genotyping precision using the Ion PGM™, the forensic sensitivity of the multiplex using dilution series, degraded DNA plus simple mixtures, and the ancestry differentiation power of the final panel design, which required substitution of three original ancestry-informative SNPs with alternatives. Fourteen populations that had not been previously analyzed were genotyped using the custom multiplex and these studies allowed assessment of genotyping performance by comparison of data across five laboratories. Results indicate a low level of genotyping error can still occur from sequence misalignment caused by homopolymeric tracts close to the target SNP, despite careful scrutiny of candidate SNPs at the design stage. Such sequence misalignment required the exclusion of component SNP rs2080161 from the Global AIM-SNPs panel. However, the overall genotyping precision and sensitivity of this custom multiplex indicates the Ion PGM™ assay for the Global AIM-SNPs is highly suitable for forensic ancestry analysis with massively parallel sequencing
Global patterns of STR sequence variation: Sequencing the CEPH human genome diversity panel for 58 forensic STRs using the Illumina ForenSeq DNA Signature Prep Kit
(Wiley, 2018-07-16) Phillips, Christopher P.; Devesse, Laurence; Ballard, David; Weert, Leanne van; Puente Vila, María del Carmen de la; Melis, Stefania; Álvarez Iglesias, Vanessa; Freire Aradas, Ana María; Oldroyd, Nicola; Holt, Cydne; Syndercombre Court, Denise; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
The 944 individuals of the CEPH human genome diversity panel (HGDP-CEPH), a standard sample set of 51 globally distributed populations, were sequenced using the Illumina ForenSeq™ DNA Signature Prep Kit. The ForenSeq™ system is a single multiplex for the MiSeq/FGx™ massively parallel sequencing instrument, comprising: amelogenin, 27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 SNPforID+Kiddlab autosomal ID-SNPs (plus optionally detected ancestry and phenotyping SNP sets). We report in detail the patterns of sequence variation observed in the repeat regions of the 58 forensic STR loci typed by the ForenSeq™ system. Sequence alleles were characterized and repeat region structures annotated by aligning the ForenSeq™ sequence output to the latest GRCh38 human reference sequence, necessitating the reversal and re-alignment of STR allele sequences reported by the Forenseq™ system in 20 of 58 STRs (plus the reverse alleles in two Y-STRs with duplicated-inverted repeat regions). Individual population sample sizes of the HGDP-CEPH panel do not allow reliable inferences to be made about levels of genetic variability in low frequency STR alleles-where particular sequence variants are found in only a few individuals; but we assessed the occurrence of both population-specific sequence variants and singleton observations; finding each of these in a sizeable proportion of HGDP-CEPH samples, with consequences for planning the co-ordinated compilation of sequence variation on a much larger scale than was required before by forensic laboratories now adopting massively parallel sequencing
A forensic multiplex of nine novel pentameric-repeat STRs
(Elsevier, 2017-04-15) Puente Vila, María del Carmen de la; Phillips, Christopher P.; Fondevila, Manuel; Gelabert Besada, Miguel; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
Pentameric-repeat short tandem repeats (STRs), consisting of loci with repeat units of five base-pairs, have the advantage of reduced stutter products compared to their tetrameric-repeat STR counterparts. This characteristic potentially helps the interpretation of mixed DNA profiles when minor component alleles may coincide with stutter peaks from the major components. To develop a simple but informative forensic multiplex with the capability to aid mixture interpretation, we designed an 11-plex assay of nine pentameric STRs new to forensic analysis plus two male- specific markers: DYS391 and the Y-Indel rs2032678 used in GlobalFiler™ (Life Technologies). East Asian-specific variation in the recently adopted Y-Indel rs2032678 is reported in this study for the first time in its forensic use as a sex marker. We estimated the levels of variation observed in the nine pentameric STRs in three of the major population groups sampled in the HGDP-CEPH human genome diversity panel: African, European and East Asian (combining individual populations as their sample sizes were too small for STR allele frequency estimations); and we include genotype data from a population sample of Northwest Spain. From this data, forensic informativeness metrics were estimated when applying the nine novel STRs in identification or kinship analyses. The assay was assessed for forensic sensitivity and ability to successfully genotype highly degraded DNA. In the profiles from the 11-plex assay we observed an average 2.15% stutter ratio in all the pentameric loci compared to 7.32% across equivalently-sized tetrameric STRs in the Promega Powerplex® ESX-17 kit
HIrisPlex-S system for eye, hair, and skin color prediction from DNA: massively parallel sequencing solutions for two common forensically used platforms
(Elsevier, 2019-08-26) Breslin, Krystal; Wills, Bailey; Ralf, Arwin; Ventayol García, Marina; Kukla Bartoszek, Magdalena; Pospiech, Ewelina; Freire Aradas, Ana María; Xavier, Catarina; Ingold, Sabrina; Puente Vila, María del Carmen de la; Gaag, Kristiaan J. van der; Herrick, Noah; Haas, Cordula; Parson, Walther; Phillips, Christopher; Sijen, Titia; Branicki, Wojciech; Walsh, Susan; Kayser, Manfred; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Forensic DNA Phenotyping (FDP) provides the ability to predict externally visible characteristics from minute amounts of crime scene DNA, which can help find unknown perpetrators who are typically unidentifiable via conventional forensic DNA profiling. Fundamental human genetics research has led to a better understanding of the specific DNA variants responsible for physical appearance characteristics, particularly eye, hair, and skin color. Recently, we introduced the HIrisPlex-S system for the simultaneous prediction of eye, hair, and skin color based on 41 DNA variants generated from two forensically validated SNaPshot multiplex assays using capillary electrophoresis (CE). Here we introduce massively parallel sequencing (MPS) solutions for the HIrisPlex-S (HPS) system on two MPS platforms commonly used in forensics, Ion Torrent and MiSeq, that cover all 41 DNA variants in a single assay, respectively. Additionally, we present the forensic developmental validation of the two HPS-MPS assays. The Ion Torrent MPS assay, based on Ion AmpliSeq technology, illustrated the successful generation of full HIrisPlex-S genotypic profiles from 100 pg of input control DNA, while the MiSeq MPS assay based on an in-house design yielded complete profiles from 250 pg of input DNA. Assessing simulated forensic casework samples such as saliva, hair (bulb), blood, semen, and low quantity touch DNA, as well as artificially damaged DNA samples, concordance testing, and samples from numerous species, all illustrated the ability of both versions of the HIrisPlex-S MPS assay to produce results that motivate forensic applications. By also providing an integrated bioinformatics analysis pipeline, MPS data can now be analyzed and a file generated for upload to the publically accessible HIrisPlex online webtool (https://hirisplex.erasmusmc.nl). In addition, we updated the website to accept VCF input data for those with genome sequence data. We thus provide a user-friendly and semi-automated MPS workflow from DNA sample to individual eye, hair, and skin color prediction probabilities. Furthermore, we present a 2-person mixture separation tool that not only assesses genotype reliability with regards genotyping confidence but also provides the most fitting mixture scenario for both minor and major contributors, including profile separation. We envision this MPS implementation of the HIrisPlex-S system for eye, hair, and skin color prediction from DNA as a starting point for further expanding MPS-based forensic DNA phenotyping. This may include the future addition of SNPs predictive for more externally visible characteristics, as well as SNPs for bio-geographic ancestry inference, provided the statistical framework for DNA prediction of these traits is in place
Development and optimization of the VISAGE basic prototype tool for forensic age estimation
(Elsevier, 2020-06-06) Heidegger, A.; Xavier, C.; Niederstätter, H.; Puente Vila, María del Carmen de la; Pospiech, E.; Pisarek, A.; Kayser, M.; Branicki, W.; Parson, W.; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
The VISAGE (VISible Attributes through GEnomics) consortium aims to develop, optimize and validate prototype tools to broaden the use of DNA intelligence methods in forensic routine laboratories. This includes age estimation based on the quantification of DNA methylation at specific CpG sites. Here, we present the VISAGE basic prototype tool for age estimation targeting 32 CpGs from five genes ELOVL2, MIR29B2CHG (herein, MIR29B2C), FHL2, TRIM59 and KLF14. The assay interrogates these well described age markers by multiplex PCR for bisulfite converted DNA and massively parallel sequencing on a MiSeq FGx instrument. We describe protocol optimizations including tests on five bisulfite conversion kits and an evaluation of the assay’s reproducibility and sensitivity with artificially methylated DNA standards. We observed robust quantification of methylation levels with a mean standard deviation of 1.4 % across ratios. Sensitivity tests showed no increase of variability down to 20 ng DNA input into bisulfite conversion with a median difference below 1.6 % between technical replicates
A compilation of tri-allelic SNPs from 1000 Genomes and use of the most polymorphic loci for a large-scale human identification panel
(Elsevier, 2020-01-02) Phillips, Christopher P.; Amigo Lechuga, Jorge; Tillmar, A. O.; Peck, M. A.; Puente Vila, María del Carmen de la; Ruiz Ramírez, Jorge; Bittner, F.; Idrizbegovic, S.; Wang, Y.; Parsons, T. J.; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
In a directed search of 1000 Genomes Phase III variation data, 271,934 tri-allelic single nucleotide polymorphisms (SNPs) were identified amongst the genotypes of 2,504 individuals from 26 populations. The majority of tri-allelic SNPs have three nucleotide substitution-based alleles at the same position, while a much smaller proportion, which we did not compile, have a nucleotide insertion/deletion plus substitution alleles. SNPs with three alleles have higher discrimination power than binary loci but keep the same characteristic of optimum amplification of the fragmented DNA found in highly degraded forensic samples. Although most of the tri-allelic SNPs identified had one or two alleles at low frequencies, often single observations, we present a full compilation of the genome positions, rs-numbers and genotypes of all tri-allelic SNPs detected by the 1000 Genomes project from the more detailed analyses it applied to Phase III sequence data. A total of 8,705 tri-allelic SNPs had overall heterozygosities (averaged across all 1000 Genomes populations) higher than the binary SNP maximum value of 0.5. Of these, 1,637 displayed the highest average heterozygosity values of 0.6-0.666. The most informative tri-allelic SNPs we identified were used to construct a large-scale human identification panel for massively parallel sequencing, designed for the identification of missing persons. The large-scale MPS identification panel comprised: 1,241 autosomal tri-allelic SNPs and 29 X tri-allelic SNPs (plus 46 microhaplotypes adapted for genotyping from reduced length sequences). Allele frequency estimates are detailed for African, European, South Asian and East Asian population groups plus the Peruvian population sampled by 1000 Genomes for the 1,270 tri-allelic SNPs of the final MPS panel. We describe the selection criteria, kinship simulation experiments and genomic analyses used to select the tri-allelic SNP components of the panel. Approximately 5 % of the tri-allelic SNPs selected for the large-scale MPS identification panel gave three-genotype patterns in single individual samples or discordant genotypes for genomic control DNAs. A likely explanation for some of these unreliably genotyped loci is that they map to multiple sites in the genome - highlighting the need for caution and detailed scrutiny of multiple-allele variant data when designing future forensic SNP panels, as such patterns can arise from common structural variation in the genome, such as segmental duplications
MAPlex - A massively parallel sequencing ancestry analysis multiplex for Asia-Pacific populations
(Elsevier, 2019-06-28) Phillips, Christopher; McNevin, Dennis B.; Kidd, Keneth K; Lagacé, R.; Wootton, S.; Puente Vila, María del Carmen de la; Freire Aradas, Ana María; Mosquera Miguel, Ana; Eduardoff, M.; Gross, T.; Dagostino, L.; Power, D.; Olson, S.; Hashiyada, Masaki; Oz, C.; Parson, Walther; Schneider, Peter M.; Laréu, Maviky Victoria; Daniel, R.; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.
Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™
(Elsevier, 2015-04-15) Eduardoff, Mayra; Santos, Carla; Puente Vila, María del Carmen de la; Gross, Theresa E.; Fondevila, María Sonia; Strobl, Christina; Sobrino, Beatriz; Ballard, David; Schneider, Peter M.; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Parson, Walther; Phillips, C.; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Next generation sequencing (NGS) offers the opportunity to analyse forensic DNA samples and obtain massively parallel coverage of targeted short sequences with the variants they carry. We evaluated the levels of sequence coverage, genotyping precision, sensitivity and mixed DNA patterns of a prototype version of the first commercial forensic NGS kit: the HID-Ion AmpliSeq™ Identity Panel with 169-markers designed for the Ion PGM™ system. Evaluations were made between three laboratories following closely matched Ion PGM™ protocols and a simple validation framework of shared DNA controls. The sequence coverage obtained was extensive for the bulk of SNPs targeted by the HID-Ion AmpliSeq™ Identity Panel. Sensitivity studies showed 90–95% of SNP genotypes could be obtained from 25 to 100pg of input DNA. Genotyping concordance tests included Coriell cell-line control DNA analyses checked against whole-genome sequencing data from 1000 Genomes and Complete Genomics, indicating a very high concordance rate of 99.8%. Discordant genotypes detected in rs1979255, rs1004357, rs938283, rs2032597 and rs2399332 indicate these loci should be excluded from the panel. Therefore, the HID-Ion AmpliSeq™ Identity Panel and Ion PGM™ system provide a sensitive and accurate forensic SNP genotyping assay. However, low-level DNA produced much more varied sequence coverage and in forensic use the Ion PGM™ system will require careful calibration of the total samples loaded per chip to preserve the genotyping reliability seen in routine forensic DNA. Furthermore, assessments of mixed DNA indicate the user’s control of sequence analysis parameter settings is necessary to ensure mixtures are detected robustly. Given the sensitivity of Ion PGM™, this aspect of forensic genotyping requires further optimisation before massively parallel sequencing is applied to routine casework.
Broadening the Applicability of a Custom Multi-Platform Panel of Microhaplotypes: Bio-Geographical Ancestry Inference and Expanded Reference Data
(Frontiers Media, 2020-10-20) Puente Vila, María del Carmen de la; Ruiz Ramírez, Jorge; Ambroa Conde, Adrián; Xavier, Catarina; Amigo, Jorge; Casares de Cal, María de los Ángeles; Gómez Tato, Antonio; Carracedo Álvarez, Ángel; Parson, Walther; Phillips, Christopher; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Departamento de Matemáticas
The development of microhaplotype (MH) panels for massively parallel sequencing (MPS) platforms is gaining increasing relevance for forensic analysis. Here, we expand the applicability of a 102 autosomal and 11 X-chromosome panel of MHs, previously validated with both MiSeq and Ion S5 MPS platforms and designed for identification purposes. We have broadened reference population data for identification purposes, including data from 240 HGDP-CEPH individuals of native populations from North Africa, the Middle East, Oceania and America. Using the enhanced population data, the panel was evaluated as a marker set for bio-geographical ancestry (BGA) inference, providing a clear differentiation of the five main continental groups of Africa, Europe, East Asia, Native America, and Oceania. An informative degree of differentiation was also achieved for the population variation encompassing North Africa, Middle East, Europe, South Asia, and East Asia. In addition, we explored the potential for individual BGA inference from simple mixed DNA, by simulation of mixed profiles followed by deconvolution of mixture components.
Determination and distribution of cannabinoids in nail and hair samples
(Oxford Academic, 2020-10-16) Cobo Golpe, María; Castro Ríos, Ana de; Cruz Landeira, Angelines; López-Rivadulla Lamas, Manuel; Lendoiro Belío, Elena; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Hair has been used for decades in toxicology as a biological matrix for long-term detection of substances. Nails are another keratinized matrix that is being studied as an alternative when hair cannot be obtained. Although cannabis is the most prevalent illicit drug in the world, cannabinoid distribution in nails compared with hair has been scarcely studied. In this work, we described two methods for the determination of cannabidiol (CBD), cannabinol (CBN) and Δ9-tetrahydrocannabinol (THC), and main metabolites of THC [11-nor-9-carboxy-THC (THCCOOH), 11-hydroxy-THC (OHTHC) and 8-β-11-dihydroxyTHC (diOHTHC)] in nail and hair samples. After an alkaline hydrolysis, samples were submitted to solid-phase extraction and analyzed by liquid chromatography with tandem mass spectrometry (LC–MS-MS). The methods were fully validated, with good linearity (r2 > 0.99) in the range of 20 to 100 to 20,000 pg/mg. No endogenous or exogenous interferences were found. Accuracy was from 99.5% to 109.8%, and imprecision was <6.9%. Ion suppression (up to −74.4%) was observed for all the analytes, except for diOHTHC at low concentrations in hair (46.1%). Extraction efficiency ranged from 21.5% to 84.5%. The methods were applied to matched nail and hair specimens from 23 cannabis users to study the incorporation and distribution of the cannabinoids into these matrices. Only CBD, CBN and THC were detected in the samples, with much higher concentrations in fingernails than in toenails and hair. Correlations between analyte concentrations in the different matrices and with reported drug consumption were studied. A preliminary cut-off for THC in toenails was calculated using the cut-off proposed by the Society of Hair Testing in hair for the identification of chronic cannabis use
A diagnostic host-specific transcriptome response for Mycoplasma pneumoniae pneumonia to guide pediatric patient treatment
(Nature Research, 2025-01-15) Viz Lasheras, Sandra; Gómez Carballa, Alberto; Bello Paderne, Xabier; Martinón Torres, Federico; Salas Ellacuriaga, Antonio; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Mycoplasma pneumoniae causes atypical pneumonia in children and young adults. Its lack of a cell wall makes it resistant to beta-lactams, which are the first-line treatment for typical pneumonia. Current diagnostic tests are time-consuming and have low specificity, leading clinicians to administer empirical antibiotics. Using a LASSO regression simulation approach and blood microarray data from 107 children with pneumonia (including 30 M. pneumoniae) we identify eight different transcriptomic signatures, ranging from 3-10 transcripts, that differentiate mycoplasma pneumonia from other bacterial/viral pneumonias with high accuracy (AUC: 0.84–0.95). Additionally, we demonstrate that existing signatures for broadly distinguishing viral/bacterial infections and viral/bacterial pneumonias are ineffective in distinguishing M. pneumoniae from viral pneumonia. The new signatures are successfully validated in an independent RNAseq cohort of children with pneumonia, demonstrating their robustness. The high sensibility of these signatures presents a valuable opportunity to guide the treatment and management of M. pneumoniae pneumonia patients.

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