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- The bigger the better? Evaluation of the value of large multi-gene panels in Portuguese cardiomyopathy genetic testingPublication . Baixia, Márcia; Sousa, Sónia; Pina, Maria J.; Silva, Raquel M; Cirnes, Luís; Canedo, Paulo; Castedo, Sérgio; Machado, José C,Introduction: Genetic testing of cardiomyopathies went through major changes in the last few years, from sequential Sanger sequencing of the most likely gene candidates, to multigene panels by NGS, with an ever increasing number of genes analyzed.
- Targeted Gene Next-Generation Sequencing Panel in Patients with Advanced Lung Adenocarcinoma: Paving the Way for Clinical ImplementationPublication . Fernandes, Maria Gabriela O.; Jacob, Maria; Martins, Natália; Moura, Conceição Souto; Guimarães, Susana; Pereira Reis, Joana; Justino, Ana; Pina, Maria João; Cirnes, Luís; Sousa, Catarina; Pinto, Josué; Marques, José Agostinho; Machado, José Carlos; Hespanhol, Venceslau; Costa, José LuisIdentification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (κ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.
- Impact of different storage conditions of formamide in the quality of sequencesPublication . Moura, Daniela; Pina, Maria João; Pereira, Cátia; Baixia, Márcia; Leal, Rafaela; Sousa, Sónia; Vieira, Filipa Quintela; Silva, Regina; Cirnes, LuísObtaining high quality sequences is essential to proper reading and interpretation. A crucial step for this goal is the denaturation of sequencing products which is usually performed using a denaturing agent such as formamide. It has been described that the reduction of formamide quality may cause irregular cytosine and guanine peaks in an electropherogram.According to manufacturers recomendations, formamide should be stored at -20ºC. Under other conditions such as exposition to light and air and temperature changes for a certain period of time, quality may decrease. Objectives: The purpose of this study was to understand which formamide storage-related variables can be responsible for decreasing sequence quality.
- Effects of different storage conditions of sequencing products with formamide in the quality of sequences.Publication . Moura, Daniela; Pina, Maria João; Pereira, Cátia; Baixia, Márcia; Leal, Rafaela; Sousa, Sónia; Vieira, Filipa Quintela; Silva, Regina; Cirnes, LuísDNA sequencing is widely used in molecular diagnosis and good sequence quality is crucial to a correct interpretation. It has been described that formamide quality decrease and sequencing reactions exposure to light, heat and/or oxygen can cause irregular peaks of cytosine and guanine in electropherograms. In a previous study, we concluded that despite the presence of this artifact when formamide is stored under non-ideal conditions, it does not significantly reduce the quality of the sequences.