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- Comparative analysis of hybrid‑SNP microarray and nanopore sequencing for detection of large‑sized copy number variants in the human genomePublication . Silva, Catarina; Ferrão, José; Marques, Bárbara; Pedro, Sónia; Correia, Hildeberto; Valente, Ana; Rodrigues, António Sebastião; Vieira, LuísBackground: Nanopore sequencing is a technology that holds great promise for identifying all types of human genome variations, particularly structural variations. In this work, we used nanopore sequencing technology to sequence 2 human cell lines at low depth of coverage to call copy number variations (CNV), and compared the results variant by variant with chromosomal microarray (CMA) results. Results: We analysed sequencing data using CuteSV and Sniffles2 variant callers, compared breakpoints based on hybrid-SNP microarray, nanopore sequencing and Sanger sequencing, and analysed CNV coverage. From a total of 48 high confidence variants (truth set), variant calling detected 79% of the truth set variants, increasing to 86% for interstitial CNV. Simultaneous use of the 2 callers slightly increased variant calling. Both callers performed better when calling CNV losses than gains. Variant sizes from CMA and nanopore sequencing showed an excellent correlation, with breakpoints determined by nanopore sequencing differing by only 20 base pairs on average from Sanger sequencing. Nanopore sequencing also revealed that four variants concealed genomic inversions undetectable by CMA. In the 10 CNV not called in nanopore sequencing, 8 showed coverage evidence of genomic loss or gain, highlighting the need to improve SV calling algorithms performance. Conclusions: Nanopore sequencing offers advantages over CMA for structural variant detection, including the identification of multiple variant types and their breakpoints with increased precision. However, further improvements in variant calling algorithms are still needed for nanopore sequencing to become a highly robust and standardized approach for a comprehensive analysis of genomic structural variation.
- Statistical analysis of longitudinal RBC omics dataPublication . Silva, Carolina; Antunes, Marília; Penque, DeborahRed blood cells (RBCs) are emerging as important modulators of the immune system. Despite evidence that alterations in RBC functionality are associated with disease severity in COVID-19 patients, there is no information regarding the impact of RBC activity on the immune response to COVID-19 vaccination. This work aims to establish an adequate methodology for the statistical analysis of longitudinal RBC metabolomics data collected during COVID-19 vaccination (n=22, 5 time points) to identify metabolites with significant changes throughout the immunization process. For the pre-treatment of the metabolomics data set, different pre-treatment methodologies comprised of imputation and normalization steps were compared to investigate which algorithm and application order was more adequate. Testing of these methods showed that normalization followed by kNN imputation using cosine distance was highlighted as the best-performing pre-treatment strategy. Following its application, generalized estimating equations (GEEs) created from the normalized data led to the identification of 30 metabolites with significant changes in concentration between different time points of COVID-19 vaccination. Significant RBC metabolites were linked to major metabolic pathways in the cells, such as the metabolism of amino acids and purines, and the transport of small molecules through the cellular membrane. Some of these metabolites were discovered to have relevant functions in the development of an effective immune response against infections, like COVID-19. The connections between these metabolites and the defense mechanisms commonly used by cells to fight viral infections offer a strong clue for the immune functions that those metabolites may have in the human body, suggesting that the RBC metabolism could play a significant part in the generation of an immune response to COVID-19 vaccination. Further work is in progress to integrate and correlate proteomic data retrieved from the same longitudinal experiment for a comprehensive depiction of the RBC function in the COVID-19 vaccine-induced immunization process.