Percorrer por autor "Machado, Miguel"
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- Avaliação do desempenho de uma core-facility de sequenciação genómica especializada em saúde públicaPublication . Vieira, Luís; Silva, Catarina; Duarte, Sílvia; Mendonça, Joana; Carpinteiro, Dina; Sampaio, Daniel A.; Ferrão, José; Santos, Daniela; Machado, Miguel; Isidro, Joana; Barreiro, Paula; Isidro, GlóriaA Unidade de Tecnologia e Inovação (UTI) do Departamento de Genética Humana foi criada em 2009 pelo despacho normativo n.º 15/2009. Apesar de estar integrada num departamento técnico científico, esta unidade constituiu-se desde logo como core-facility de sequenciação genómica do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA). Este papel envolve uma gestão contínua de prioridades dos serviços a prestar aos utilizadores, no âmbito da resposta a diferentes problemas de saúde pública, aliada a uma preocupação permanente com a qualidade dos resultados e os tempos de resposta. Neste trabalho, apresentamos os resultados da avaliação do desempenho da UTI, desde a introdução da tecnologia de Next-Generation Sequencing (NGS) em 2013, em termos de: (i) métricas de produção da Unidade, (ii) impacto dos resultados publicados no âmbito de colaborações científicas com os grupos de investigação do INSA ou de entidades externas e de (iii) avaliação dos serviços através de um inquérito dirigido aos utilizadores. Até final de 2021, o número de ensaios de NGS e de citações dos trabalhos publicados cresceram, por ano, 39% e 61%, respetivamente. Os utilizadores avaliaram de forma muito positiva os serviços prestados pela UTI em 2021. Globalmente, estes resultados demonstram que o modelo de trabalho de "core- -facility" exercido pela UTI é uma mais-valia na resposta aos problemas da saúde pública em Portugal.
- Genetic variants in the IFNGR2 locus associated with severe chronic Q feverPublication . David, Susana; Castro, Liliana; Duarte, Elsa; Gaspar, Ulisses; Rodrigues, Maria Rosário da Costa; Cueto-Rojo, Maria Vanessa; Mendonça, Joana; Ferrão, José; Machado, Miguel; Poças, José; Lavinha, João; Vieira, Luís; Santos, Ana Sofia; ElsevierQ fever is a highly contagious zoonosis capable of causing large outbreaks of important health and economic consequences. Host genetic factors are believed to influence the development of severe chronic Q fever following the infection by the etiological agent, Coxiella burnetii. Targetted next generation sequencing (NGS) was performed in a case-control genetic association study on 53 confirmed Q fever cases, including 38 compatible with acute and 15 with chronic disease, and 29 samples from the general Portuguese population. Four SNPs in the IFNGR2 locus, rs78407108 G > A, rs17879956 C > T, rs7277167 C > T, and rs9974603 C > A, showed a statistically significant association to chronic Q fever, resisting the Bonferroni correction. These belonged to haplotypes significantly associated with chronic Q fever. The individual SNPs are referenced in the GTEx database as possible eQTLs. Given the direct bearing of IFNGR2 on IFN-γ signaling, the possible involvement of the associated variants with higher IFNGR2 expression could be in line with observations suggesting that IFN-γ production in chronic Q fever patients is significantly higher than in healthy controls. Further investigations are required to clarify the role of IFNGR2 signaling in association with chronic Q fever.
- The Technology and Innovation Unit of the National Institute of Health: A sequencing and bioinformatics core facility specializing in public health genomicsPublication . Silva, Catarina; Ataíde Sampaio, Daniel; Mendonça, Joana; Carpinteiro, Dina; Duarte, Sílvia; Barreiro, Paula; Isidro, Joana; Machado, Miguel; Vieira, LuísThe National Institute of Health (INSA) has a long tradition in investigating the molecular etiology of genetic and complex diseases. These activities greatly benefit from centralized sequencing services provided by the Technology and Innovation Unit (UTI). Its mission is to perform sequencing and genotyping assays in the framework of research, diagnosis and epidemiological surveillance, as well as to implement data analysis pipelines for the study of human gene variants. The equipment portfolio includes a NextSeq 550, a MiSeq, two 3500 AB Genetic Analyzers, a Fragment Analyzer and a 7500 Real-time PCR system. UTI provides results for average of 36.000 sequencing/fragment samples per year. The team has already performed >300 small genome, amplicon, gene panel (including clinical exome), 16S rRNA gene and RNA/microRNA next-generation sequencing assays for INSA and for several Universities in the scope of scientific collaborations. Technical procedures are conducted under a quality control system that includes external quality assessment for next-generation sequencing/Sanger sequencing and ISO 15189 accreditation for Sanger sequencing. UTI plays a key role in public health genomics, providing state-of-the-art equipment, centralized resources, technical expertise and short response times.
- The Technology and Innovation Unit of the National Institute of Health: a sequencing and bioinformatics core facility specializing in public health genomicsPublication . Silva, Catarina; Ataíde Sampaio, Daniel; Mendonça, Joana; Carpinteiro, Dina; Duarte, Sílvia; Barreiro, Paula; Isidro, Joana; Machado, Miguel; Vieira, LuísThe National Institute of Health (INSA) is the state laboratory in the health sector. INSA has a long tradition in investigating the molecular etiology of genetic and complex diseases and in the identification of pathogenic organisms responsible for disease outbreaks and environmental imbalances. These activities benefit greatly from the existing centralized sequencing services provided by the Technology and Innovation Unit (UTI). Its mission includes performing sequencing and genotyping assays in the framework of research, diagnosis and epidemiological surveillance, as well as implementing data analysis pipelines for the study of variation in human genes. The equipment portfolio includes two next-generation sequencers and two capillary electrophoresis instruments for Sanger sequencing/fragment analysis, that altogether process an average of 36.000 samples/year. The team performed over 300 next-generation sequencing workflows for small genomes, amplicons, gene panels, clinical exome, 16S rRNA gene and RNA/microRNAs. Standard of operation procedures are conducted by trained technicians under a quality control system that includes external quality assessment and ISO 15189 accreditation. UTI plays a key role in public health genomics, providing state-of-the-art equipment, centralized resources, technical expertise and short response times for public health problems.
- Whole human genome 5’-mC methylation analysis using long read nanopore sequencingPublication . Silva, Catarina; Machado, Miguel; Ferrão, José; Sebastião Rodrigues, António; Vieira, LuísMethylation microarray and bisulphite sequencing are often used to study 5'-methylcytosine (5'-mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustworthy results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulphite and/or the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5'-mC since it allows sequencing of native DNA molecules, whereas the long reads produced by this technology help to increase the resolution of those genomic regions. In this work, we show that nanopore sequencing with 10X coverage depth, using DNA from a human cell line, produces 5'-mC methylation frequencies consistent with those obtained by 450k microarray, digital restriction enzyme analysis of methylation, and reduced representation bisulphite sequencing. High correlation between methylation frequencies obtained by nanopore sequencing and the other methodologies was also noticeable in either low or high GC content regions, including CpG islands and transcription start sites. We also showed that a minimum of five reads per CpG yields strong correlations (>0.89) in replicate nanopore sequencing runs and an almost uniform linearity of the methylation frequency variation between zero and one. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns based on genomic annotations of CpG regions. These results demonstrate that nanopore sequencing is a fast, robust, and reliable approach to the study of 5'-mC in the human genome with low coverage depth.