Investigating Highly Variable Genes in Single-cell RNA-seq Data across Multiple Cell Types and Conditions

The peripheral blood immune cell (PBMC) samples were collected from patients infected with dengue virus (DENV) at four time points: two and one day(s) before defervescence (febrile phase), at defervescence (critical phase), and two-week convalescence. The raw and filtered matrix files were generated using CellRanger version 3.0.2 (10x Genomics, USA) with the reference human genome GRCh38 1.2.0. Potential contamination of ambient RNAs was corrected using SoupX. Low quality cells, including cells expressing mitochondrial genes higher than 10% and doublets/multiplets, were excluded using Seurat and doubletFinder, respectively. The individual samples were then integrated using the SCTransform method with 3,000 gene features. Principal component analysis (PCA) and clustering were performed with the Louvain algorithm applying multi-level refinement algorithm. The gene expression level of each cell was normalized using the LogNormalize method in Seurat. Cell types were annotated using the canonical marker genes described in the original paper, see related link below.