diff --git a/.gitattributes b/.gitattributes new file mode 100644 index 0000000..07fe41c --- /dev/null +++ b/.gitattributes @@ -0,0 +1,2 @@ +# GitHub syntax highlighting +pixi.lock linguist-language=YAML linguist-generated=true diff --git a/.gitignore b/.gitignore new file mode 100644 index 0000000..096b5eb --- /dev/null +++ b/.gitignore @@ -0,0 +1,3 @@ +# pixi environments +.pixi +*.egg-info diff --git a/README.md b/README.md index 608a98a..62781ad 100644 --- a/README.md +++ b/README.md @@ -1,2 +1,11 @@ -# This repository contains the code used for our manuscript: -https://www.biorxiv.org/content/10.1101/2023.08.24.554683 +# G1/S Boundary Activates Interferon and Inflammatory Response Genes + +## [Manuscript](https://www.biorxiv.org/content/10.1101/2023.08.24.554683) + +## Running + +### RNA-seq + +```sh +pixi run plots +``` diff --git a/RNA-seq/01_STAR_align.sh b/RNA-seq/01_STAR_align.sh old mode 100644 new mode 100755 index c1fa727..c0fdc4c --- a/RNA-seq/01_STAR_align.sh +++ b/RNA-seq/01_STAR_align.sh @@ -1,6 +1,7 @@ #! /bin/bash # load STAR -module load star/2.7.3a +# TODO if Ganymede +# module load star/2.7.3a # Read file names while IFS=',' read -ra array; do diff --git a/RNA-seq/02_samtools_filter.sh b/RNA-seq/02_samtools_filter.sh old mode 100644 new mode 100755 diff --git a/RNA-seq/03_bam_index.sh b/RNA-seq/03_bam_index.sh old mode 100644 new mode 100755 diff --git a/RNA-seq/04_DEG_analysis.Rmd b/RNA-seq/04_DEG_analysis.Rmd index 840fd50..7b69416 100644 --- a/RNA-seq/04_DEG_analysis.Rmd +++ b/RNA-seq/04_DEG_analysis.Rmd @@ -14,50 +14,19 @@ knitr::opts_chunk$set(echo = TRUE) ```{r packages, echo=TRUE,eval=FALSE,cache=FALSE} # install & load packages -#install.packages("dplyr") library(dplyr) #install.packages("DT") -#install.packages("tidyr") library(tidyr) -#install.packages("ggplot2") library(ggplot2) -#install.packages("magrittr") library(magrittr) -#install.packages("devtools") library(devtools) - -#source("https://bioconductor.org/biocLite.R") - -# Needed for mac and Linux only -# BiocManager::install("Rsubread") +# Bioconductor library(Rsubread) - -# BiocManager::install("Rsamtools") library(Rsamtools) -# BiocManager::install("GenomicAlignments") -# BiocManager::install("TxDb.Hsapiens.UCSC.hg38.knownGene") library(TxDb.Hsapiens.UCSC.hg38.knownGene) -# BiocManager::install("soGGi") -# BiocManager::install("rtracklayer") -# BiocManager::install("ChIPQC") -# BiocManager::install("ChIPseeker") -# BiocManager::install("rGREAT") -# BiocManager::install("limma") -# BiocManager::install("DESeq2") library(DESeq2) -# BiocManager::install("tracktables") -# BiocManager::install("clusterProfiler") -# BiocManager::install("org.Mm.eg.db") -# BiocManager::install("MotifDb") -# BiocManager::install("Biostrings") -# BiocManager::install("edgeR") library(edgeR) -# BiocManager::install("BSgenome.Hsapiens.UCSC.hg38") - -# Finally we need development version of soGGi (named here 1.10.4) -# not version on Bioconductor (1.10.0) -# devtools::install_github("ThomasCarroll/soGGi") - sessionInfo() +sessionInfo() ``` ```{r, echo=TRUE,eval=FALSE,cache=FALSE} diff --git a/RNA-seq/05_plots.R b/RNA-seq/05_plots.R old mode 100644 new mode 100755 index 7635c01..9d42297 --- a/RNA-seq/05_plots.R +++ b/RNA-seq/05_plots.R @@ -1,20 +1,19 @@ +#!/usr/bin/env Rscript + #### Create plots for the paper #### # packages library(edgeR) library(DESeq2) -#BiocManager::install("reshape2") library(reshape2) library(ggplot2) library(dplyr) library(tidyr) library(RColorBrewer) library(Cairo) -#BiocManager::install("ggrepel") library(ggrepel) -#BiocManager::install("ggrastr") library(ggrastr) library(pheatmap) -library("stringr") +library(stringr) ##################################################### # set the colors for samples hex_cols <- c("#1b9e77", "#d95f02", "#7570b3", "#e7298a") @@ -22,10 +21,12 @@ hex_cols <- c("#1b9e77", "#d95f02", "#7570b3", "#e7298a") # PCA plot # GM12878 # get the data +# FIXME This hard-coded path will fail load(file="~/project/Gozde_data/RNA-seq/R_objs/GM_counts_keep.RData") # create group names for GM samples Group_GM <- factor(c("GM_DU","GM_DI", "GM_AU", "GM_AI", "GM_DU", "GM_DI", "GM_AU", "GM_AI", "GM_DU", "GM_DI", "GM_AU", "GM_AI")) colnames(GM_counts_keep) <- Group_GM +# FIXME This hard-coded path will fail load(file="~/project/Gozde_data/RNA-seq/R_objs/GM_RNA_DDS.RData") #normalize the reads @@ -46,10 +47,12 @@ ggsave(pca_gm, filename= "~/Gozde_data/RNA-seq/files/PCA_GM.pdf", device = cairo # JURKAT # get the data +# FIXME This hard-coded path will fail load("~/project/Gozde_data/RNA-seq/R_objs/JU_counts_keep.RData") # create group names for JU samples Group_JU <- factor(c("JU_DU","JU_DI", "JU_AU", "JU_AI", "JU_DU", "JU_DI", "JU_AU", "JU_DI", "JU_AI", "JU_AU", "JU_AI", "JU_DU")) colnames(JU_counts_keep) <- Group_JU +# FIXME This hard-coded path will fail load(file="~/project/Gozde_data/RNA-seq/R_objs/JU_RNA_DDS.RData") #normalize the reads @@ -149,6 +152,7 @@ dev.off() ###################################################### rm(list = ls()) +# TODO There might be more packages in here library(dplyr) library(ggpubr) library(Seurat) @@ -157,9 +161,11 @@ library(destiny) library(ggrastr) library(DESeq2) library(edgeR) +# FIXME This hard-coded path will fail source("~/onlybiohpc/pr3/OUR_DATA/utility_functions.R") # Load dataset +# FIXME This hard-coded path will fail load(file="~/project/Gozde_data/RNA-seq/R_objs/GM_RNA_DDS.RData") # Normalize the reads @@ -208,6 +214,7 @@ dev.off() # Overlaps - AI-DU and AU-DU +# TODO Add to pixi library(VennDiagram) pdf('GM_AIDU_AUDU_OVERLAP.pdf') @@ -317,6 +324,7 @@ ggscatter(toplot, x = 'log10FDR', y = 'Description', color = 'red', size = 'Odds dev.off() ## HEATMAPS ## +# FIXME This hard-coded path will fail load("~/project/Gozde_data/RNA-seq/R_objs/GM_counts_keep.RData") GM_counts_norm = log2(cpm(GM_counts_keep) + 1) @@ -563,6 +571,7 @@ dev.off() ## GM vs JU # Load datasets +# FIXME This hard-coded path will fail load(file="~/project/Gozde_data/RNA-seq/R_objs/JU_counts_keep.RData") load(file="~/project/Gozde_data/RNA-seq/R_objs/GM_counts_keep.RData") @@ -609,6 +618,7 @@ rio::export(DU_GM_JU, file="~/project/Gozde_data/RNA-seq/files/DU_GM_vs_JU.xlsx" ## GM and JU markers in AU vs DU +# FIXME This hard-coded path will fail load(file="~/project/Gozde_data/RNA-seq/R_objs/GM_RNA_DDS.RData") load(file="~/project/Gozde_data/RNA-seq/R_objs/JU_RNA_DDS.RData") @@ -627,6 +637,7 @@ ju_au_du_up = ju_au_du[ju_au_du$log2FoldChange > 0.6 & ju_au_du$padj < 0.05,] %> #length(intersect(gm_ju_down, gm_au_du_up)) / length(union(gm_ju_down, gm_au_du_up)) +# TODO Add to pixi library(GeneOverlap) gm_ju_list = list(gm_ju_up, gm_ju_down) au_du_list = list(gm_au_du_up, ju_au_du_up) @@ -707,6 +718,7 @@ dev.off() # Overlaps - AI-DU and AU-DU +# TODO Add to pixi library(VennDiagram) pdf('JU_AIDU_AUDU_OVERLAP.pdf') @@ -730,6 +742,7 @@ draw.pairwise.venn(area1 = length(c(ai_up, ai_down)), dev.off() # Overlap with GM +# FIXME This hard-coded path will fail load(file="~/project/Gozde_data/RNA-seq/R_objs/GM_RNA_DDS.RData") GM_AU_DU = results(GM_RNA_DDS, c("Group_GM", "GM_AU", "GM_DU"), format = "DataFrame") GM_AU_DU = GM_AU_DU[!(is.na(GM_AU_DU$padj)),] @@ -747,6 +760,7 @@ draw.pairwise.venn(area1 = length(c(au_up, au_down)), cat.cex = 2, cat.fontface = 'bold', margin = 0.13) dev.off() +# TODO Add to pixi library(GeneOverlap) resgom = newGOM(list(c(au_up, au_down)), list(c(au_up_gm, au_down_gm)), genome.size = nrow(GM_AU_DU)) pvalmat = getMatrix(resgom, name="pval") @@ -870,6 +884,7 @@ dev.off() ## HEATMAPS ## +# FIXME This hard-coded path will fail load("~/project/Gozde_data/RNA-seq/R_objs/JU_counts_keep.RData") JU_counts_norm = log2(cpm(JU_counts_keep) + 1) diff --git a/pixi.lock b/pixi.lock new file mode 100644 index 0000000..1eda9d8 --- /dev/null +++ b/pixi.lock @@ -0,0 +1,6134 @@ +version: 5 +environments: + default: + channels: + - 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libgcc-ng >=12 + - libstdcxx-ng >=12 + - libzlib >=1.2.13,<2.0.0a0 + license: BSD-3-Clause + license_family: BSD + size: 554846 + timestamp: 1714722996770 diff --git a/pixi.toml b/pixi.toml new file mode 100644 index 0000000..4fd897e --- /dev/null +++ b/pixi.toml @@ -0,0 +1,38 @@ +[project] +authors = ["Edmund Miller "] +channels = ["conda-forge", "bioconda"] +description = "Add a short description here" +name = "IFN_heterogeneity" +platforms = ["linux-64"] +version = "0.1.0" + +[feature.rnaseq.dependencies] +star = "2.7.3a.*" +samtools = "1.10.*" +r-dplyr = ">=1.0.10,<2" +r-tidyr = ">=1.2.1,<2" +r-ggplot2 = ">=3.3.6,<4" +r-magrittr = ">=2.0.3,<3" +r-devtools = ">=2.4.4,<3" +bioconductor-rsubread = ">=2.4.3,<3" +bioconductor-rsamtools = ">=2.6.0,<3" +"bioconductor-txdb.hsapiens.ucsc.hg38.knowngene" = ">=3.10.0,<4" +bioconductor-deseq2 = ">=1.30.1,<2" +bioconductor-edger = ">=3.32.1,<4" +bioconductor-soggi = ">=1.10.4,<2" +r-markdown = ">=1.7,<2" +r-reshape2 = ">=1.4.4,<2" +r-ggrepel = ">=0.9.3,<0.10" +r-ggrastr = ">=1.0.2,<2" +r-pheatmap = ">=1.0.12,<2" +r-stringr = ">=1.5.0,<2" + +[feature.rnaseq.tasks] +align = { cmd = "01_STAR_align.sh", cwd = "RNA-seq" } +filter = { cmd = "02_samtools_filter.sh", depends-on = ["align"], cwd = "RNA-seq" } +index = { cmd = "03_bam_index.sh", depends-on = ["filter"], cwd = "RNA-seq" } +DEG_analysis = { cmd = "Rscript -e \"rmarkdown::render('04_DEG_analysis.Rmd')\"", depends-on = ["filter", "index"], cwd = "RNA-seq" } # TODO outputs = ["report.html"] +plots = { cmd = "Rscript -e \"rmarkdown::render('04_DEG_analysis.Rmd')\"", depends-on = ["DEG_analysis", "index"], cwd = "RNA-seq", outputs = ["BoxPlot_MotifGenes_common_JU.pdf", "PCA_GM.pdf", "PCA_JU.pdf", "Boxplot_IFNB1_GM.pdf"] } + +[environments] +rnaseq = ["rnaseq"]