mitra-frn / sc-TE-RNA

sc-TE-RNA

Correlation of myeloid-derived suppressor cell expansion with upregulated transposable elements in severe COVID-19 un-veiled in single-cell RNA sequencing reanalysis

Description of scripts used in the pipeline

Part I: Align reads to referance genome

bash Script	alinerscripts.sh
Input	human/hg38 genome sequence
	annotation of referance genome on GTF format
	cDNAfragmentSequence.fastq and CellBarcodeUMIsequence.fastq for each sample cDNAfragment example and CellBarcodeUMI example
	v3 cell barcode whitelist file 3M-february-2018.txt
	repeatmasker.bed
Output	count matrixes on h5ad format
Dependencies	STAR 2.7.10a,scTE(needs python >=3.6),bedtools
Summary	Genome indexes were generated and Patients' paired-end FASTQ files were mapped to them.transposable element were quantified using scTE package algorithm.

Part II: Preprocessing, analysis, and exploration of scRNA-seq data

R Script	Seuratscripts.R
Input	count matrixes on h5ad format examples of count matrix
Output	Defferential expression tables for each cluster in compares to other clusters
Dependencies	zellkonverter,SingleCellExperiment,Seurat
Summary	count matrixes were preprocessed(ig.,filtering,normalizaton).The bach effects between samples were removed and all-samples of each condition(ie,healthy,mild and severe conditions) were integrated.After scaling data, PCA was performed and The KNN graph was conducted based on the PCA-reduced data, and unsupervised clustering was performed.Differential expression analysis was conducted based on the 'MAST' method.

Part III: Scoring pathways and correlation test

R Script	scoringscripts.R
Input	seurat-object of samples example of seurat metadata
	gene-sets of pathways example
Output	correlation tables and relating p.adjvalue
Dependencies	Seurat,psych
Summary	Average expression levels of gene-sets pathways in neutrophil clusters and upregulated genes in these clusters(logfc>0.7 and p.adj<0.05) were calculated and the correlation between these pathways and upregulated genes in neutrophil clusters was evaluated.