Antonio Gómez-Tato

Inferring ancestral origin using a single multiplex assay of ancestry-informative marker SNPs

Tests that infer the ancestral origin of a DNA sample have considerable potential in the development of forensic tools that can help to guide crime investigation. We have developed a single-tube 34-plex SNP assay for the assignment of ancestral origin by choosing ancestry-informative markers (AIMs) exhibiting highly contrasting allele frequency distributions between the three major population-groups. To predict ancestral origin from the profiles obtained, a classification algorithm was developed based on maximum likelihood. Sampling of two populations each from African, European and East Asian groups provided training sets for the algorithm and this was tested using the CEPH Human Genome Diversity Panel. We detected negligible theoretical and practical error for assignments to one of the three groups analyzed with consistently high classification probabilities, even when using reduced subsets of SNPs. This study shows that by choosing SNPs exhibiting marked allele frequency differences between population-groups a practical forensic test for assigning the most likely ancestry can be achieved from a single multiplexed assay.

Analysis of global variability in 15 established and 5 new European Standard Set (ESS) STRs using the CEPH human genome diversity panel

The CEPH human genome diversity cell line panel (CEPH-HGDP) of 51 globally distributed populations was used to analyze patterns of variability in 20 core human identification STRs. The markers typed comprised the 15 STRs of Identifiler, one of the most widely used forensic STR multiplexes, plus five recently introduced European Standard Set (ESS) STRs: D1S1656, D2S441, D10S1248, D12S391 and D22S1045. From the genotypes obtained for the ESS STRs we identified rare, intermediate or off-ladder alleles that had not been previously reported for these loci. Examples of novel ESS STR alleles found were characterized by sequence analysis. This revealed extensive repeat structure variation in three ESS STRs, with D12S391 showing particularly high variability for tandem runs of AGAT and AGAC repeat units. The global geographic distribution of the CEPH panel samples gave an opportunity to study in detail the extent of substructure shown by the 20 STRs amongst populations and between their parent population groups. An assessment was made of the forensic informativeness of the new ESS STRs compared to the loci they will replace: CSF1PO, D5S818, D7S820, D13S317 and TPOX, with results showing a clear enhancement of discrimination power using multiplexes that genotype the new ESS loci. We also measured the ability of Identifiler and ESS STRs to infer the ancestry of the CEPH-HGDP samples and demonstrate that forensic STRs in large multiplexes have the potential to differentiate the major population groups but only with sufficient reliability when used with other ancestry-informative markers such as single nucleotide polymorphisms. Finally we checked for possible association by linkage between the two ESS multiplex STRs closely positioned on chromosome-12: vWA and D12S391 by examining paired genotypes from the complete CEPH data set.

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 ancestry-informative-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 naïve 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.

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens

BackgroundThe turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis.ResultsA total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value ≤ 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot.ConclusionA collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.

Further development of forensic eye color predictive tests.

In forensic analysis predictive tests for external visible characteristics (or EVCs), including inference of iris color, represent a potentially useful tool to guide criminal investigations. Two recent studies, both focused on forensic testing, have analyzed single nucleotide polymorphism (SNP) genotypes underlying common eye color variation (Mengel-From et al., Forensic Sci. Int. Genet. 4:323 and Walsh et al., Forensic Sci. Int. Genet. 5:170). Each study arrived at different recommendations for eye color predictive tests aiming to type the most closely associated SNPs, although both confirmed rs12913832 in HERC2 as the key predictor, widely recognized as the most strongly associated marker with blue and brown iris colors. Differences between these two studies in identification of other eye color predictors may partly arise from varying approaches to assigning phenotypes, notably those not unequivocally blue or dark brown and therefore occupying an intermediate iris color continuum. We have developed two single base extension assays typing 37 SNPs in pigmentation-associated genes to study SNP-genotype based prediction of eye, skin, and hair color variation. These assays were used to test the performance of different sets of eye color predictors in 416 subjects from six populations of north and south Europe. The presence of a complex and continuous range of intermediate phenotypes distinct from blue and brown eye colors was confirmed by establishing eye color populations compared to genetic clusters defined using Structure software. Our study explored the effect of an expanded SNP combination beyond six markers has on the ability to predict eye color in a forensic test without extending the SNP assay excessively - thus maintaining a balance between the tests predictive value and an ability to reliably type challenging DNA with a multiplex of manageable size. Our evaluation used AUC analysis (area under the receiver operating characteristic curves) and naïve Bayesian likelihood-based classification approaches. To provide flexibility in SNP-based eye color predictive tests in forensic applications we modified an online Bayesian classifier, originally developed for genetic ancestry analysis, to provide a straightforward system to assign eye color likelihoods from a SNP profile combining additional informative markers from the predictors analyzed by our study plus those of Walsh and Mengel-From. Two advantages of the online classifier is the ability to submit incomplete SNP profiles, a common occurrence when typing challenging DNA, and the ability to handle physically linked SNPs showing independent effect, by allowing the user to input frequencies from SNP pairs or larger combinations. This system was used to include the submission of frequency data for the SNP pair rs12913832 and rs1129038: indicated by our study to be the two SNPs most closely associated to eye color.

Molecular Characterization of Circulating Tumor Cells in Human Metastatic Colorectal Cancer

Metastatic colorectal cancer (mCRC) relies on the detachment of aggressive malignant cells from the primary tumor into the bloodstream and, concordantly, the presence of these Circulating Tumor Cells (CTC) is associated with a poor prognosis. In this work, the molecular characterization of CTC from mCRC patients was approached, with the aim of understanding their biology and improving their clinical utility in the management of colorectal cancer patients. For this, EpCAM-based immunoisolation of CTC was combined with whole transcriptome amplification and hybridization onto cDNA microarrays. Gene expression data from mCRC patients, once the background of unspecific immunoisolation from a group of controls had been subtracted, resulted in 410 genes that characterized the CTC population. Bioinformatics were used for the biological interpretation of the data, revealing that CTC are characterized by genes related to cell movement and adhesion, cell death and proliferation, and cell signalling and interaction. RTqPCR on an independent series of mCRC patients and controls was used for the validation of a number of genes related to the main cellular functions characterizing the CTC population. Comparison between primary carcinomas and lung and liver metastases further involved the CTC-genes in the promotion of metastasis. Moreover, the correlation of CTC-gene expression with clinical parameters demonstrated detection and prognosis significance. In conclusion, the molecular characterization of CTC from mCRC patients and the identification of diagnostic and prognostic biomarkers represent an innovative and promising approach in the clinical management of this type of patients.

Whole Genome Sequencing of Turbot (Scophthalmus maximus; Pleuronectiformes): A Fish Adapted to Demersal Life

The turbot is a flatfish (Pleuronectiformes) with increasing commercial value, which has prompted active genomic research aimed at more efficient selection. Here we present the sequence and annotation of the turbot genome, which represents a milestone for both boosting breeding programmes and ascertaining the origin and diversification of flatfish. We compare the turbot genome with model fish genomes to investigate teleost chromosome evolution. We observe a conserved macrosyntenic pattern within Percomorpha and identify large syntenic blocks within the turbot genome related to the teleost genome duplication. We identify gene family expansions and positive selection of genes associated with vision and metabolism of membrane lipids, which suggests adaptation to demersal lifestyle and to cold temperatures, respectively. Our data indicate a quick evolution and diversification of flatfish to adapt to benthic life and provide clues for understanding their controversial origin. Moreover, we investigate the genomic architecture of growth, sex determination and disease resistance, key traits for understanding local adaptation and boosting turbot production, by mapping candidate genes and previously reported quantitative trait loci. The genomic architecture of these productive traits has allowed the identification of candidate genes and enriched pathways that may represent useful information for future marker-assisted selection in turbot.

A combined strategy involving Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot (Scophthalmus maximus)

BackgroundGenomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry.ResultsExpressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immune-related tissues after several parasitic challenges. The resulting database (“Turbot 2 database”) was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences (“Turbot 3 database”), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50–90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified.ConclusionsThe combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs.

Automated extraction improves multiplex molecular detection of infection in septic patients.

Sepsis is one of the leading causes of morbidity and mortality in hospitalized patients worldwide. Molecular technologies for rapid detection of microorganisms in patients with sepsis have only recently become available. LightCycler SeptiFast test Mgrade (Roche Diagnostics GmbH) is a multiplex PCR analysis able to detect DNA of the 25 most frequent pathogens in bloodstream infections. The time and labor saved while avoiding excessive laboratory manipulation is the rationale for selecting the automated MagNA Pure compact nucleic acid isolation kit-I (Roche Applied Science, GmbH) as an alternative to conventional SeptiFast extraction. For the purposes of this study, we evaluate extraction in order to demonstrate the feasibility of automation. Finally, a prospective observational study was done using 106 clinical samples obtained from 76 patients in our ICU. Both extraction methods were used in parallel to test the samples. When molecular detection test results using both manual and automated extraction were compared with the data from blood cultures obtained at the same time, the results show that SeptiFast with the alternative MagNA Pure compact extraction not only shortens the complete workflow to 3.57 hrs., but also increases sensitivity of the molecular assay for detecting infection as defined by positive blood culture confirmation.

An Expressed Sequence Tag (EST)-enriched genetic map of turbot (Scophthalmus maximus): a useful framework for comparative genomics across model and farmed teleosts

BackgroundThe turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies.ResultsA consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups.ConclusionsThe new gene-enriched high-resolution turbot map represents a useful genomic tool for QTL identification, positional cloning strategies, and future genome assembling. This map showed large synteny conservation against model teleost genomes. Comparative genomics and data mining from landmarks will provide straightforward access to candidate genes, which will be the basis for genetic breeding programs and evolutionary studies in this species.