scRNAseq_DENVtimecourse

Description

This repository contains the required codes to perform scRNA-sequencing analysis in our publication entitled "Single-cell Temporal Analysis of Natural Human Dengue Virus Infection Reveals Expansion of Skin Homing Lymphocyte Subsets One Day before Defervescence"

Data

Data was deposited in the ArrayExpress repository under the accession number E-MTAB-9467

Raw data processing

Sequenced data was analysed using CellRanger (v.3.0.2) and reference human genome GRCh38 1.2.0

cellranger count --id = <sample_id> \
                 --transcriptome = <refdata-cellranger-GRCh38-1.2.0> \
                 --fastqs = <fastq_path> \
                 --sample = <sample_name> \
                 --chemistry = SC3Pv2 \
                 --expect-cells = 5000

R scripts

R scripts for data analysis in this publication including

• Part01 : Pre-processing steps and quality control of each sample
  The standard pipeline from Seurat (v.3.1.2) was applied for data normalisation, clustering and dimensionality reduction. To remove the potential contamination of ambient RNAs and doublets, SoupX (v.1.4.5) and DoubletFinder (v.2.0.3) were performed before integrating the data.

• Part02 : Data normalisation and integration (Figure 1B)
  The SCTransform from Seurat (v.3.1.2) was used for normalisation prior to integration.

• Part03 : Highly variable genes (HVGs) and biological process (BP) analyses (Figure 2A-C)
  The Principal Component Analysis (PCA) was performed (Figure 2A). The union of the top 500 genes from PC1 and PC2 (HVGs) were then used to construct the heatmap (Figure 2B). The HVG and BP analyses of each immune cell type (Figure 2C) were used the same Rscript, except PCA and HVGs were constructed using the integrated object from each cell type.

• Part04 : Trajectory and pseudotime analyses (Figure 3E)

Environments and Dependencies

• Cell Ranger (v.3.0.2) (Docker is available)

R studio v.4.0.2 and the packages used in this publication (Docker is available)

• SoupX (v.1.4.5)
• DoubletFinder (v.2.0.3)
• Seurat (v.3.1.2)
• Monocle3 (v.0.2.3.0)
• gProfiler2 (v.0.1.9)

Data analyses were performed on Ubuntu 16.04.6 LTS