Single Cell RNA Sequencing with Hybrid Pipeline

Published as Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium, Langlois et al. 2020

The journal link: PLOS journal link

The 10x Chromium Single Cell sequencing raw data of experimental samples alignment using Cell Ranger Count (v3.0.1)

The alignment is done in a hybrid manner with human+SARS-CoV-2 viral genome reference (Homo_sapiens.GRCh38; MN985325)

The hybrid genome bash script: hybrid_pipeline_bash_script.sh

The raw data can be found in NCBI GEO website: GSE157526

This NCBI GEO website includes raw count matrix for each experimental data set.

The overall hybrid pipeline bash steps:

• Concatenating nucleotide and assembly's annotation and gtf files
• Creating Cell Ranger reference
• Alignment and Count using Cell Ranger Count for each of the sample (the filter criteria for empty droplets are minimum 1000 genes per cell)

Analysis Method for the raw count matrix was done using R (v3.6.3) with Seurat (v2.3.4)

The Seurat hybrid pipeline: hybrid_pipeline_seurat_R_script.R

The CoV2 cells analysis (an extension from the Seurat hybrid pipeline: CoV2_cells_analysis_seurat_R_script.R

The overall hybrid pipeline Seurat steps:

• QC and filtering on metadata (filter for genes with minimum three cells and for mitochondria genes percentage less than 30%)
• Data normalization, scaling, and detection of variable genes
• Dimensionality Reduction using PCA and clustering analysis (mitochondria and viral genes excluded)
• Find all gene markers and key genes results visualization

The overall CoV2 cells analysis steps:

• Subset data for lower threshold of 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1% CoV2 cells
• Plot TSNE dimensionality reduction with specific threshold virus cells highlighted

Analysis Method for integration was done using R (v4.0.2) with Seurat (v3.2.2)

The Seurat integration pipeline: integration_pipeline_seurat_v3_R_script.R

This pipeline includes CoV2 (definition >0.001% CoV2) and IFN cell analysis.

The overall integration pipeline Seurat steps:

• Update Seurat object to v3
• Merge and integrate anchorset
• Normalization and scalling
• Dimensionality reduction: PCA and UMAP resolution 0.15
• Plot UMAP cell highlight for CoV2 (>0.001% cadetblue color) and IFN (red color)
• Plot FeatureScatter normalized CoV2 and IFN for sample 2 (24h) and 5 (48h)