Building a custom large-scale panel of novel microhaplotypes for forensic identification using MiSeq and Ion S5 massively parallel sequencing systems

A large number of new microhaplotype loci were identified in the human genome by applying a directed search with selection criteria emphasizing short haplotype length (<120 nucleotides) and maximum levels of polymorphism in the composite SNPs. From these searches, 107 autosomal microhaplotypes and 11 X chromosome microhaplotypes were selected, with well-spaced autosomal positions to ensure their independence in relationship tests. The 118 microhaplotypes were assembled into a single multiplex assay for the analysis of forensic DNA with massively parallel sequencing (MPS). A single AmpliSeq-adapted primer set was made for Illumina MiSeq and Thermo Fisher Ion S5 MPS platforms and the performance of the assay was comprehensively evaluated in both systems. Five microhaplotypes showed critical sequencing failures in both MPS platforms and were removed, while a further 13 required manual checks and the application of sequence quality thresholds in one or both systems to ensure the successful analysis of low-level DNA in these loci. The targeting of short microhaplotype spans during marker selection, with an average length of 51 nucleotides in the 118 loci, led to a high level of sensitivity for the panel when sequencing the very degraded DNA typically encountered in forensic casework and the identification of missing personsThe studies reported and authors MdlP, CP, MVL are supported by MAPA: Multiple Allele Polymorphism Analysis (BIO2016-78525-R), a research project funded by the Spanish Research State Agency (AEI), and co-financed with ERDF funds. MdlP is supported by a postdoctoral fellowship awarded by the Consellería de Cultura, Educación e Ordenación Universitaria and the Consellería de Economía, Emprego e Industria of the Xunta de Galicia (ED481B 2017/088). The studies reported have received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 740580 within the framework of the Visible Attributes Through Genomics (VISAGE) Project and ConsortiumS

Ancestry Analysis in the 11-M Madrid Bomb Attack Investigation

The 11-M Madrid commuter train bombings of 2004 constituted the second biggest terrorist attack to occur in Europe after Lockerbie, while the subsequent investigation became the most complex and wide-ranging forensic case in Spain. Standard short tandem repeat (STR) profiling of 600 exhibits left certain key incriminatory samples unmatched to any of the apprehended suspects. A judicial order to perform analyses of unmatched samples to differentiate European and North African ancestry became a critical part of the investigation and was instigated to help refine the search for further suspects. Although mitochondrial DNA (mtDNA) and Y-chromosome markers routinely demonstrate informative geographic differentiation, the populations compared in this analysis were known to show a proportion of shared mtDNA and Y haplotypes as a result of recent gene-flow across the western Mediterranean, while any two loci can be unrepresentative of the ancestry of an individual as a whole. We based our principal analysis on a validated 34plex autosomal ancestryinformative-marker single nucleotide polymorphism (AIM-SNP) assay to make an assignment of ancestry for DNA from seven unmatched case samples including a handprint from a bag containing undetonated explosives together with personal items recovered from various locations in Madrid associated with the suspects. To assess marker informativeness before genotyping, we predicted the probable classification success for the 34plex assay with standard error estimators for a naı¨ve Bayesian classifier using Moroccan and Spanish training sets (each n = 48). Once misclassification error was found to be sufficiently low, genotyping yielded seven near-complete profiles (33 of 34 AIM-SNPs) that in four cases gave probabilities providing a clear assignment of ancestry. One of the suspects predicted to be North African by AIM-SNP analysis of DNA from a toothbrush was identified late in the investigation as Algerian in origin. The results achieved illustrate the benefit of adding specialized marker sets to provide enhanced scope and power to an already highly effective system of DNA analysis for forensic identification.European Commission GROWTH program, SNPforID project, contract G6RD-CT-2002-00844 to CP. Xunta de Galicia, Spain: Fund PGIDTIT06PXIB228195PR and Ministerio de Educación y Ciencia, Spain: project BIO2006-06178 to MVL. Fundación de Investigación Médica Mutua Madrileña, Spain: 2006/CL370 and 2008/CL444 to AS. Continued development of the work and its application to forensic analysis is being funded by Allelyus, Santiago de Compostela, SpainS

Epigenetic age estimation in saliva and in buccal cells

Age estimation based on epigenetic markers is a DNA intelligence tool with the potential to provide relevant information for criminal investigations, as well as to improve the inference of age-dependent physical characteristics such as male pattern baldness or hair color. Age prediction models have been developed based on different tissues, including saliva and buccal cells, which show different methylation patterns as they are composed of different cell populations. On many occasions in a criminal investigation, the origin of a sample or the proportion of tissues is not known with certainty, for example the provenance of cigarette butts, so use of combined models can provide lower prediction errors. In the present study, two tissue-specific and seven age-correlated CpG sites were selected from publicly available data from the Illumina HumanMethylation 450 BeadChip and bibliographic searches, to help build a tissue-dependent, and an age-prediction model, respectively. For the development of both models, a total of 184 samples (N = 91 saliva and N = 93 buccal cells) ranging from 21 to 86 years old were used. Validation of the models was performed using either k-fold cross-validation and an additional set of 184 samples (N = 93 saliva and N = 91 buccal cells, 21–86 years old). The tissue prediction model was developed using two CpG sites (HUNK and RUNX1) based on logistic regression that produced a correct classification rate for saliva and buccal swab samples of 88.59 % for the training set, and 83.69 % for the testing set. Despite these high success rates, a combined age prediction model was developed covering both saliva and buccal cells, using seven CpG sites (cg10501210, LHFPL4, ELOVL2, PDE4C, HOXC4, OTUD7A and EDARADD) based on multivariate quantile regression giving a median absolute error (MAE): ± 3.54 years and a correct classification rate ( %CP±PI) of 76.08 % for the training set, and an MAE of ± 3.66 years and a %CP±PI of 71.19 % for the testing set. The addition of tissue-of origin as a co-variate to the model was assessed, but no improvement was detected in age predictions. Finally, considering the limitations usually faced by forensic DNA analyses, the robustness of the model and the minimum recommended amount of input DNA for bisulfite conversion were evaluated, considering up to 10 ng of genomic DNA for reproducible results. The final multivariate quantile regression age predictor based on the models we developed has been placed in the open-access Snipper forensic classification websiteThis project was funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (Modalidade B, ED481B 2018/010) by a postdoctorate grant awarded to AFA. MVL is supported by the Ministerio de Educación, Cultura y Ciencia, Spain (PID2019-107876RB-I00).M.d.l.P. is supported by a post-doctorate grant funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (ED481D-2021-008). J.R. is supported by the “Programa de axudas á etapa predoutoral” funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (ED481A-2020/039)S

Forensic evaluation of the Asia Pacific ancestry-informative MAPlex assay

DNA intelligence, and particularly the inference of biogeographical ancestry (BGA) is increasing in interest, and relevance within the forensic genetics community. The majority of current MPS-based forensic ancestry-informative assays focus on the differentiation of major global populations. The recently published MAPlex (Multiplex for the Asia Pacific) panel contains 144 SNPs and 20 microhaplotypes and aims to improve the differentiation of populations in the Asia Pacific region. This study reports the first forensic evaluation of the MAPlex panel using AmpliSeq technology and Ion S5 sequencing. This study reports on the overall performance of MAPlex including the assay’s sequence coverage distribution and stability, baseline noise and description of problematic SNPs. Dilution series, artificially degraded and mixed DNA samples were also analysed to evaluate the sensitivity of the panel with challenging or compromised forensic samples. As the first panel to combine biallelic SNPs, multiple-allele SNPs and microhaplotypes, the MAPlex assay demonstrated an enhanced capacity for mixture detection, not easily performed with common binary SNPs. This performance evaluation indicates that MAPlex is a robust, stable and highly sensitive assay that is applicable to forensic casework for the prediction of BGAMdlP is supported by a postdoctoral fellowship awarded by the Consellería de Cultura, Educación e Ordenación Universitaria and the Consellería de Economía, Emprego e Industria from Xunta de Galicia (Modalidade A, ED481B 2017/088). CP, AFA, AMM, MdlP, MVL are supported by MAPA, Multiple Allele Polymorphism Analysis (BIO2016-78525-R), a research project funded by the Spanish Research State Agency (AEI), and co-financed with ERDF funds. AFA is supported by a post-doctorate grant funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (Modalidade B, ED481B 2018/010). The 1000 Genomes high coverage sequence data were generated at the New York Genome Center with funds provided by NHGRI Grant 3UM1HG008901-03S1S

Phenotyping the ancient world: the physical appearance and ancestry of very degraded samples from a chalcolithic human remains

The genetic study of ancient samples is quite similar to a forensic critical sample analysis with an unknown origin. In both cases, it is not possible to compare the genetic information with other family members, being almost impossible to achieve the individual identification. The prediction of externally visible characteristics (EVC) of an individual and his biogeographical ancestry could definitely be a crucial contribution in a forensic casework. Therefore, the aim of the present work was the molecular study of a very critical sample, a Chalcolithic (3480 ± 30 YBP) individual found in Asturias, Northern Iberia, intending to discover a possible geographical ancestry for these remains, and the inference of a group of feasible EVCs (hair, skin and iris pigmentation). Given that ancient DNA is often highly damaged, two different methodologies were used in order to determine the biogeographical ancestry of the individual: mitochondrial DNA (HVR-I and -II) and Single Nucleotide Polymorphisms typing. Despite the antiquity of the samples, the genetic information recovered proved of great value. We could determine that the individual had a European ancestry, blond hair, light skin color and brown eyes. Such outcome reveals that it is possible to obtain not only biogeographical but also phenotypic information from a very critical sample

Humanized medium (h7H) allows long-term primary follicular thyroid cultures from human normal thyroid, benign neoplasm, and cancer

Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid cancer lines were found to be heavily contaminated by other sources, and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor.|The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype.|Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed.|We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas.|Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.Ministerio de Ciencia e InnovaciónInstituto de Salud Carlos IIIXunta de GaliciaFondo Social Europeo of the European Communit

Mitochondrial echoes of first settlement and genetic continuity in El Salvador

Background: From Paleo-Indian times to recent historical episodes, the Mesoamerican isthmus played an important role in the distribution and patterns of variability all around the double American continent. However, the amount of genetic information currently available on Central American continental populations is very scarce. In order to shed light on the role of Mesoamerica in the peopling of the New World, the present study focuses on the analysis of the mtDNA variation in a population sample from El Salvador. Methodology/Principal Findings: We have carried out DNA sequencing of the entire control region of the mitochondrial DNA (mtDNA) genome in 90 individuals from El Salvador. We have also compiled more than 3,985 control region profiles from the public domain and the literature in order to carry out inter-population comparisons. The results reveal a predominant Native American component in this region: by far, the most prevalent mtDNA haplogroup in this country (at ~90%) is A2, in contrast with other North, Meso- and South American populations. Haplogroup A2 shows a star-like phylogeny and is very diverse with a substantial proportion of mtDNAs (45%; sequence range 16090–16365) still unobserved in other American populations. Two different Bayesian approaches used to estimate admixture proportions in El Salvador shows that the majority of the mtDNAs observed come from North America. A preliminary founder analysis indicates that the settlement of El Salvador occurred about 13,400±5,200 Y.B.P.. The founder age of A2 in El Salvador is close to the overall age of A2 in America, which suggests that the colonization of this region occurred within a few thousand years of the initial expansion into the Americas. Conclusions/Significance: As a whole, the results are compatible with the hypothesis that today's A2 variability in El Salvador represents to a large extent the indigenous component of the region. Concordant with this hypothesis is also the observation of a very limited contribution from European and African women (~5%). This implies that the Atlantic slave trade had a very small demographic impact in El Salvador in contrast to its transformation of the gene pool in neighbouring populations from the Caribbean facade

Genetics Unveil the Genealogical Ancestry and Physical Appearance of an Unknown Historical Figure: Lady Leonor of Castile (Spain) (1256–1275)

Through this study, it has been possible to establish an accurate prediction of the physical characteristics, biogeographical origin, and genealogical ancestry of a previously obscured historical figure: The Princess Lady Leonor of Castile (1256–1275), one of the legitimate daughters of the Spanish King Alfonso X “The Wise”. The genetic analysis of External Visible Characteristics in the mummified remains attributed to this Princess has allowed determining her origin by mitochondrial and nuclear DNA analysis, and her physical appearance for hair, eyes, and skin color by autosomal SNPs. The results show that the mummified remains correspond to a young European woman with black hair, green-hazel eyes, and white skin. Her physical appearance has not been possible to be compared with any pictorial source, but the biogeographical analysis results are consistent with the historiographic genealogical information

Genetics unveil the genealogical ancestry and physical appearance of an unknown historical figure: lady Leonor of Castile (Spain) (1256–1275)

2023 Descuento MDPIThrough this study, it has been possible to establish an accurate prediction of the physical characteristics, biogeographical origin, and genealogical ancestry of a previously obscured historical figure: The Princess Lady Leonor of Castile (1256–1275), one of the legitimate daughters of the Spanish King Alfonso X “The Wise”. The genetic analysis of External Visible Characteristics in the mummified remains attributed to this Princess has allowed determining her origin by mitochondrial and nuclear DNA analysis, and her physical appearance for hair, eyes, and skin color by autosomal SNPs. The results show that the mummified remains correspond to a young European woman with black hair, green-hazel eyes, and white skin. Her physical appearance has not been possible to be compared with any pictorial source, but the biogeographical analysis results are consistent with the historiographic genealogical information.Depto. de Medicina Legal, Psiquiatría y PatologíaFac. de MedicinaTRUEpubDescuento UC