PirbrightBAL-yang

This repository contains analysis scripts for analysing porcine BAL scRNAseq data in Pirbright Institute. Results are now published in The Journal of Immunology, titled; 'Porcine respiratory coronavirus as a model for acute respiratory disease: mechanisms of different infection outcomes'. https://doi.org/10.1093/jimmun/vkaf066

Original unprocessed data can be downloaded from GEO(GSE288145). https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE288145

To replicate the analysis performed in the published paper, download the fastq.gz files from GEO(GSE288145) and perform alignment/counting using cellranger count (cellranger-7.1.0) using default parameters and Sus scrofa genome (genome assembly 11.1, Ensembl release 108) to regenerate files used in the original analysis. Example cellranger commands can be found in '/cluster scripts'.

Directory should look like this for each sample folder:

• sample1 folder
  • molecule_info.h5
  • raw_feature_bc_matrix.h5 also available on GEO
  • raw_feature_bc_matrix folder
    • barcodes.tsv.gz
    • features.tsv.gz
    • matrix.mtx.gz

Scripts in this directory are run in the following order:

1. processing_all_lily.Rmd Reads in cellranger output (see above) and performs initial filtering and QC.
2. clust_lily.Rmd Clustering and cluster inspection.
3. differential_lily.Rmd Cell type annotation followed by an differential expression analysis utilising a pseudobulk approach.
4. advanced_analysis.Rmd GO and KEGG enrichment of the identified differentially expressed genes.

shiny_app/data.rds to use for data exploration in the shiny app shiny_app/app.R is generated by clustering all.Rmd (unannotated data).

1. demux/muci_demux.sh
2. count/muci_count_11.sh, count/muci_count_12.sh, count/muci_count_13.sh
3. html/muci_html_preprocess.sh
4. html/muci_html_cluster.sh
5. html/html_differential.sh
6. html/html_advanced_analysis.sh

sbatch demux/muci_demux.sh

sbatch count/muci_count_11.sh

sbatch count/muci_count_12.sh

sbatch count/muci_count_13.sh

sbatch html/muci_html_preprocess.sh

sbatch html/muci_html_cluster.sh

sbatch html/html_differential.sh

sbatch -w compute001 html/html_advanced_analysis.sh