RESUMEN
Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.
Asunto(s)
Linfocitos B , Tonsila Palatina , Humanos , Adulto , Linfocitos B/metabolismoRESUMEN
BACKGROUND: Modern horses represent heterogeneous populations specifically selected for appearance and performance. Genomic regions under high selective pressure show characteristic runs of homozygosity (ROH) which represent a low genetic diversity. This study aims at detecting the number and functional distribution of ROHs in different horse populations using next generation sequencing data. METHODS: Next generation sequencing was performed for two Sorraia, one Dülmen Horse, one Arabian, one Saxon-Thuringian Heavy Warmblood, one Thoroughbred and four Hanoverian. After quality control reads were mapped to the reference genome EquCab2.70. ROH detection was performed using PLINK, version 1.07 for a trimmed dataset with 11,325,777 SNPs and a mean read depth of 12. Stretches with homozygous genotypes of >40 kb as well as >400 kb were defined as ROHs. SNPs within consensus ROHs were tested for neutrality. Functional classification was done for genes annotated within ROHs using PANTHER gene list analysis and functional variants were tested for their distribution among breed or non-breed groups. RESULTS: ROH detection was performed using whole genome sequences of ten horses of six populations representing various breed types and non-breed horses. In total, an average number of 3492 ROHs were detected in windows of a minimum of 50 consecutive homozygous SNPs and an average number of 292 ROHs in windows of 500 consecutive homozygous SNPs. Functional analyses of private ROHs in each horse revealed a high frequency of genes affecting cellular, metabolic, developmental, immune system and reproduction processes. In non-breed horses, 198 ROHs in 50-SNP windows and seven ROHs in 500-SNP windows showed an enrichment of genes involved in reproduction, embryonic development, energy metabolism, muscle and cardiac development whereas all seven breed horses revealed only three common ROHs in 50-SNP windows harboring the fertility-related gene YES1. In the Hanoverian, a total of 18 private ROHs could be shown to be located in the region of genes potentially involved in neurologic control, signaling, glycogen balance and reproduction. Comparative analysis of homozygous stretches common in all ten horses displayed three ROHs which were all located in the region of KITLG, the ligand of KIT known to be involved in melanogenesis, haematopoiesis and gametogenesis. CONCLUSIONS: The results of this study give a comprehensive insight into the frequency and number of ROHs in various horses and their potential influence on population diversity and selection pressures. Comparisons of breed and non-breed horses suggest a significant artificial as well as natural selection pressure on reproduction performance in all types of horse populations.
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Cruzamiento , Caballos/genética , Reproducción/genética , Selección Genética , Animales , Genómica , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Fenotipo , Polimorfismo de Nucleótido SimpleRESUMEN
Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Here we describe a method to target, multiplex and sequence at high coverage full-length human mitochondrial genomes as native single-molecules, utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks, that define the mtDNA beginning and end of the sequencing reads, as barcodes, we achieve high demultiplexing specificity and delineation of the full-length of the mtDNA, regardless of the structural variant pattern. The long-read sequencing data is analysed with a pipeline where our custom-developed software, baldur, efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a tool for studying mtDNA variation and will accelerate mitochondrial research.
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Genoma Mitocondrial , ADN Mitocondrial/genética , Desoxirribonucleasa I/genética , Genoma Humano/genética , Genoma Mitocondrial/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Nucleótidos , ARN , Análisis de Secuencia de ADN/métodosRESUMEN
Benzo[b]cyclohexa-2,4-dien-1-one (4) and benzo[b]cyclohexa-2,5-dien-1-one (5), the two most stable keto tautomers of 1-naphthol (1), were generated in aqueous solution by Norrish Type II fission of 4- and 2-phenacyl-1-tetralone, respectively, and the pH-rate profiles of their enolization were measured by flash photolysis. Several isotopic exchange rates of 1 were measured in aqueous acid to determine the corresponding rate constants of ketonization. The resulting equilibrium constants for enolization are pKE(4) = -7.1 and pKE(5) = -6.2. The acidity constants of the carbon acids 4 and 5, pKa(4) = 2.1 and pKa(5) = 3.0, were then obtained from a thermodynamic cycle using pKa(1) = 9.25.
RESUMEN
Most available protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative, or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by polymerase chain reaction (PCR) with one of the two primers being biotinylated. The PCR-generated template is rendered single stranded and a pyrosequencing primer is annealed to analyze quantitatively CpGs within 120 bases. Advantages of the pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain high reproducibility and avoid random amplification. The required DNA amount can be provided by an individual sample or a pool of samples to rapidly investigate the presence of variable DNA methylation patterns. The use of pools and serial pyrosequencing, that is, the successive use of several pyrosequencing primers on the same DNA template, significantly reduces cost, labor, and analysis time as well as saving precious DNA samples for the analysis of gene-specific DNA methylation patterns.
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Metilación de ADN , Análisis de Secuencia de ADN/métodos , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/genética , Islas de CpG , ADN/química , ADN/genética , ADN/aislamiento & purificación , Cartilla de ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Genes p16 , Genómica , Humanos , Neoplasias Hepáticas/química , Neoplasias Hepáticas/genética , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
Causes of high mortality of prostate cancer in men of African ancestry living in the French West Indies are still debated, between suspicions of environmental factors and genetic susceptibility. We report an integrated genomic study of 25 tumour tissues from radical prostatectomy of aggressive (defined by International Society of Urological Pathology ≥3) prostate cancer patients (10 African Caribbean and 15 French Caucasian) using single nucleotide polymorphism arrays, whole-genome sequencing, and RNA sequencing. The results show that African Caribbean tumours are characterised by a more frequent deletion at 1q41-43 encompassing the DNA repair gene PARP1, and a higher proportion of intrachromosomal rearrangements including duplications associated with CDK12 truncating mutations. Transcriptome analyses show an overexpression of genes related to androgen receptor activity in African Caribbean tumours, and of PVT1, a long non-coding RNA located at 8q24 that confirms the strong involvement of this region in prostate tumours from men of African ancestry. Patient summary: Mortality of prostate cancer is higher in African Caribbean men than in French Caucasian men. Specificities of the former could be explained by genomic events linked with key genes such as DNA damage pathway genes PARP1, CDK12, and the oncogenic long non-coding RNA gene PVT1 at the 8q24 prostate cancer susceptibility locus.
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Población Negra/genética , Neoplasias de la Próstata/genética , Población Blanca/genética , Región del Caribe/etnología , Humanos , Masculino , Mutación , Polimorfismo de Nucleótido Simple , Prostatectomía , Neoplasias de la Próstata/etnología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Secuenciación Completa del GenomaRESUMEN
Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.
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Islas de CpG , Proteínas de Unión al ADN/metabolismo , Impresión Genómica , Proteínas/genética , ARN no Traducido/genética , Proteínas Represoras/metabolismo , Sitios de Unión , Factor de Unión a CCCTC , Metilación de ADN , Femenino , Expresión Génica , Genotipo , Humanos , Recién Nacido , Patrón de Herencia , Factor II del Crecimiento Similar a la Insulina , Masculino , Modelos Genéticos , Linaje , Placenta/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas/metabolismo , ARN Largo no Codificante , ARN no Traducido/metabolismo , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Many protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative and/or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by PCR with one of the two primers being biotinylated. The PCR generated template is rendered single-stranded and a pyrosequencing primer is annealed to analyze quantitatively cytosine methylation. In comparative studies, pyrosequencing has been shown to be among the most accurate and reproducible technologies for locus-specific DNA methylation analyses and has become a widely used tool for the validation of DNA methylation changes identified in genome-wide studies as well as for locus-specific analyses with clinical impact such as methylation analysis of the MGMT promoter. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity.
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Metilación de ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Nucleótidos/química , Análisis de Secuencia de ADN/métodos , Proteínas Supresoras de Tumor/genética , Islas de CpG , ADN de Cadena Simple/química , Humanos , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , SulfitosRESUMEN
Aberrant DNA methylation patterns have been identified in a variety of human diseases, particularly cancer. Pyrosequencing has evolved in recent years as a sensitive and accurate method for the analysis and quantification of the degree of DNA methylation in specific target regions. However, the number of candidate genes that can be analyzed in clinical specimens is often restricted by the limited amount of sample available. Here, we present a novel screening approach that enables the rapid identification of differentially methylated regions such as promoters by pyrosequencing of etiologically homogeneous sample pools after bisulfite treatment. We exemplify its use by the analysis of five genes (CDKN2A, GSTP1, MLH1, IGF2, and CTNNB1) involved in the pathogenesis of human hepatocellular carcinoma using pools stratified for different parameters of clinical importance. Results were confirmed by the individual analysis of the samples. The screening identified all genes displaying differential methylation successfully, and no false positives occurred. Quantitative comparison of the pools and the samples in the pool analyzed individually showed a deviation of approximately 1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material.
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Carcinoma Hepatocelular/genética , Metilación de ADN , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN/métodos , Islas de CpG , ADN de Neoplasias/genética , Humanos , Tamaño de la MuestraRESUMEN
As the sequence of the human genome is now nearly finished, genome research turns to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the pathogenesis of various diseases. The real-time luminometric detection of pyrophosphate release upon nucleotide incorporation in the Pyrosequencing technology is ideally suited for the simultaneous analysis and quantification of the methylation degree of several CpG positions in close proximity. We developed and improved this analysis to obtain reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 80 nt. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity, which may be used as DNA methylation markers.
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Metilación de ADN , Difosfatos/metabolismo , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , ADN/genética , ADN/aislamiento & purificación , Exones/genética , Genoma Humano/genética , Antígenos HLA/análisis , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/análisis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Sulfitos/metabolismo , Moldes GenéticosRESUMEN
BACKGROUND: Changes in microbial community composition in the lung of patients suffering from moderate to severe COPD have been well documented. However, knowledge about specific microbiome structures in the human lung associated with CT defined abnormalities is limited. METHODS: Bacterial community composition derived from brush samples from lungs of 16 patients suffering from different CT defined subtypes of COPD and 9 healthy subjects was analyzed using a cultivation independent barcoding approach applying 454-pyrosequencing of 16S rRNA gene fragment amplicons. RESULTS: We could show that bacterial community composition in patients with changes in CT (either airway or emphysema type changes, designated as severe subtypes) was different from community composition in lungs of patients without visible changes in CT as well as from healthy subjects (designated as mild COPD subtype and control group) (PC1, Padj = 0.002). Higher abundance of Prevotella in samples from patients with mild COPD subtype and from controls and of Streptococcus in the severe subtype cases mainly contributed to the separation of bacterial communities of subjects. No significant effects of treatment with inhaled glucocorticoids on bacterial community composition were detected within COPD cases with and without abnormalities in CT in PCoA. Co-occurrence analysis suggests the presence of networks of co-occurring bacteria. Four communities of positively correlated bacteria were revealed. The microbial communities can clearly be distinguished by their associations with the CT defined disease phenotype. CONCLUSION: Our findings indicate that CT detectable structural changes in the lung of COPD patients, which we termed severe subtypes, are associated with alterations in bacterial communities, which may induce further changes in the interaction between microbes and host cells. This might result in a changed interplay with the host immune system.
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Bacterias/clasificación , Pulmón/microbiología , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico por imagen , Análisis de Secuencia de ADN/métodos , Tomografía Computarizada por Rayos X/métodos , Anciano , Bacterias/genética , Bacterias/aislamiento & purificación , Código de Barras del ADN Taxonómico/métodos , ADN Bacteriano/genética , ADN Ribosómico/genética , Femenino , Humanos , Pulmón/diagnóstico por imagen , Masculino , Microbiota , Persona de Mediana Edad , Prevotella/clasificación , Prevotella/genética , Prevotella/aislamiento & purificación , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , ARN Ribosómico 16S/genética , Streptococcus/clasificación , Streptococcus/genética , Streptococcus/aislamiento & purificaciónRESUMEN
The analysis of deoxyribose nucleic acid (DNA) by mass spectrometry (MS) has evolved to where it can be used to analyze most known types of DNA and ribose nucleic acid (RNA) situations. It can efficiently deal with the analysis of DNA polymorphisms, sequences, haplotypes, human leukocyte antigen (HLA) typing, DNA methylation and RNA expression. Implementations of MS for these forms of DNA analyses are reviewed. The use of DNA analysis by MS is compared with competing technologies. Finally, an overview is given of worthwhile applications where the know-how gained so far could be used for future developments.
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ADN/análisis , Espectrometría de Masas , Animales , Metilación de ADN , Expresión Génica , Genómica , Genotipo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Espectrometría de Masas/historia , Espectrometría de Masas/tendencias , Polimorfismo de Nucleótido Simple , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/historia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/tendenciasRESUMEN
As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R(2) >or= 0.99 and standard deviation around 2% for the different positions. The limit of detection was approximately 5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.
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Islas de CpG/genética , Metilación de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Bases , ADN/química , ADN/genética , ADN/metabolismo , ADN de Neoplasias/química , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Factor VIII/genética , Genotipo , Gutatión-S-Transferasa pi , Glutatión Transferasa/genética , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodosRESUMEN
Reconstruction of haplotypes, or the allelic phase, of single nucleotide polymorphisms (SNPs) is a key component of studies aimed at the identification and dissection of genetic factors involved in complex genetic traits. In humans, this often involves investigation of SNPs in case/control or other cohorts in which the haplotypes can only be partially inferred from genotypes by statistical approaches with resulting loss of power. Moreover, alternative statistical methodologies can lead to different evaluations of the most probable haplotypes present, and different haplotype frequency estimates when data are ambiguous. Given the cost and complexity of SNP studies, a robust and easy-to-use molecular technique that allows haplotypes to be determined directly from individual DNA samples would have wide applicability. Here, we present a reliable, automated and high-throughput method for molecular haplotyping in 2 kb, and potentially longer, sequence segments that is based on the physical determination of the phase of SNP alleles on either of the individual paternal haploids. We demonstrate that molecular haplotyping with this technique is not more complicated than SNP genotyping when implemented by matrix-assisted laser desorption/ionisation mass spectrometry, and we also show that the method can be applied using other DNA variation detection platforms. Molecular haplotyping is illustrated on the well-described beta(2)-adrenergic receptor gene.
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Haplotipos/genética , Polimorfismo de Nucleótido Simple/genética , Alelos , Secuencia de Bases , ADN/química , ADN/genética , Electroforesis en Gel de Agar , Frecuencia de los Genes , Genotipo , Humanos , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
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Adaptación Fisiológica , Peces Planos/genética , Genoma , Animales , Evolución Molecular , Proteínas de Peces/genética , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
HER2-positive breast cancer has long proven to be a clinically distinct class of breast cancers for which several targeted therapies are now available. However, resistance to the treatment associated with specific gene expressions or mutations has been observed, revealing the underlying diversity of these cancers. Therefore, understanding the full extent of the HER2-positive disease heterogeneity still remains challenging. Here we carry out an in-depth genomic characterization of 64 HER2-positive breast tumour genomes that exhibit four subgroups, based on the expression data, with distinctive genomic features in terms of somatic mutations, copy-number changes or structural variations. The results suggest that, despite being clinically defined by a specific gene amplification, HER2-positive tumours melt into the whole luminal-basal breast cancer spectrum rather than standing apart. The results also lead to a refined ERBB2 amplicon of 106 kb and show that several cases of amplifications are compatible with a breakage-fusion-bridge mechanism.
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Neoplasias de la Mama/genética , Receptor ErbB-2/metabolismo , Neoplasias de la Mama/metabolismo , Variaciones en el Número de Copia de ADN , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Amplificación de Genes , Perfilación de la Expresión Génica , Humanos , Mutación , Polimorfismo de Nucleótido Simple , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Transcriptoma , Secuenciación Completa del GenomaRESUMEN
Medicine and healthcare are undergoing profound changes. Whole-genome sequencing and high-resolution imaging technologies are key drivers of this rapid and crucial transformation. Technological innovation combined with automation and miniaturization has triggered an explosion in data production that will soon reach exabyte proportions. How are we going to deal with this exponential increase in data production? The potential of "big data" for improving health is enormous but, at the same time, we face a wide range of challenges to overcome urgently. Europe is very proud of its cultural diversity; however, exploitation of the data made available through advances in genomic medicine, imaging, and a wide range of mobile health applications or connected devices is hampered by numerous historical, technical, legal, and political barriers. European health systems and databases are diverse and fragmented. There is a lack of harmonization of data formats, processing, analysis, and data transfer, which leads to incompatibilities and lost opportunities. Legal frameworks for data sharing are evolving. Clinicians, researchers, and citizens need improved methods, tools, and training to generate, analyze, and query data effectively. Addressing these barriers will contribute to creating the European Single Market for health, which will improve health and healthcare for all Europeans.
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Investigación Biomédica/legislación & jurisprudencia , Bases de Datos Factuales/normas , Unión Europea/organización & administración , Investigación Biomédica/normas , Bases de Datos Factuales/legislación & jurisprudencia , Implementación de Plan de Salud , Humanos , Difusión de la Información/legislación & jurisprudenciaRESUMEN
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become one of the most powerful and widely applied technologies for SNP scoring and determination of allele frequencies in the post-genome sequencing era. Although different strategies for allele discrimination combined with MALDI were devised, in practice only primer extension methods are nowadays routinely used. This combination enables the rapid, quantitative, and direct detection of several genetic markers simultaneously in a broad variety of biological samples. In the field of molecular diagnostics, MALDI has been applied to the discovery of genetic markers, that are associated with a phenotype like a disease susceptibility or drug response, as well as an alternative means for diagnostic testing of a range of diseases for which the responsible mutations are already known. It is one of the first techniques with which whole genome scans based on single nucleotide polymorphisms were carried out. It is equally well suited for pathogen identification and the detection of emerging mutant strains as well as for the characterization of the genetic identity and quantitative trait loci mapping in farm animals. MALDI can also be used as a detection platform for a range of novel applications that are more demanding than standard SNP genotyping such as mutation/polymorphism discovery, molecular haplotyping, analysis of DNA methylation, and expression profiling. This review gives an introduction to the application of mass spectrometry for DNA analysis, and provides an overview of most studies using SNPs as genetic markers and MALDI mass spectrometric detection that are related to clinical applications and molecular diagnostics. Further, it aims to show specialized applications that might lead to diagnostic applications in the future. It does not speculate on whether this methodology will ever reach the diagnostic market.
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Pruebas Genéticas/métodos , Polimorfismo de Nucleótido Simple , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Secuencia de Bases , Marcadores Genéticos , Genotipo , Humanos , Sensibilidad y EspecificidadRESUMEN
The last decade has seen an increased demand for high-throughput DNA analysis. This is mainly due to the human genome sequencing project that is now completed. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was pinpointed early on as a technology that could be of great use for sequence variation analysis in the post-genome sequencing era. Applications developed first on this platform were for SNP genotyping. Several strategies for allele-discrimination (hybridization, cleavage, ligation, and primer extension) were combined with MALDI-TOF mass spectrometric detection. Nowadays, in practice, only primer extension methods are applied for large-scale SNP genotyping studies with MALDI-TOF detection. Problems surrounding the integration of SNP genotyping by MALDI-TOF mass spectrometry at high throughput are largely mastered now. Mass spectrometry geared presentations at the HUGO Mutation Detection Meeting in Palm Cove, Australia almost exclusively focused on novel applications that go beyond standard SNP genotyping. These applications are more demanding in terms of chemistry and molecular biology. Molecular haplotyping, expression profiling, DNA methylation analysis, and mutation detection are now being demonstrated.