2.4. Pathway Analysis
Bioproject data was obtained from PRJNA615032 bioproject trancriptome data, which includes lung biopsies from SARS-CoV-2-infected patients and healthy volunteers as well as mock and SARS-CoV-2-transfected NHEB and A549 cell lines. The data have been deposited with links to BioProject accession number PRJNA615032 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/). All the selected data were reanalysed at the Rosalind bioinformatics server. Data analysis was performed according to 1.5 fold change between untransfected and transfected cell lines in a data pool calculation for both cell lines at p < 0.05 significance level. Data was analyzed by Rosalind (https://rosalind.onramp.bio/), with a HyperScale architecture developed by OnRamp BioInformatics, Inc. (San Diego, CA, USA). Reads were trimmed using cutadapt [25]. Quality scores were assessed using FastQC [26]. Reads were aligned to the Homo sapiens genome built by GRCh38 using STAR [27]. Individual sample reads were quantified using HTseq [28] and normalized via relative log expression (RLE) using DESeq2 R library [29]. Read distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step using RSeQC [30]. DEseq2 was also used to calculate fold changes and p-values and perform optional covariate correction. Clustering of genes for the final heatmap of differentially expressed genes was done using the PAM (partitioning around medoids) method using the fpc R library (https://cran.r-project.org/web/packages/fpc/index.html). Hypergeometric distribution was used to analyze the enrichment of pathways, gene ontology, domain structure, and other ontologies. The topGO R library [31], was used to determine local similarities and dependencies between GO terms in order to perform Elim pruning correction. Several database sources were referenced for enrichment analysis, including Interpro [32], NCBI [33], MSigDB [34,35], REACTOME [36], and WikiPathways [37]. Enrichment was calculated relative to a set of background genes relevant for the experiment.