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01_MakePhenoCov.R

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+library(tidyr, lib="/apps/source/R/3.6.3/lib64/R/library/")
+library(fansi, lib="/apps/source/R/3.6.3/lib64/R/library/")
+library(dplyr, lib="/apps/source/R/3.6.3/lib64/R/library/")
+
+###module load R/3.6.3
+###inputs are:
+###intcol
+###isolate
+
+args <- commandArgs(trailingOnly=TRUE)
+
+id_map <- read.csv("../SecondRound_JatinFeatures/wgs.map.ModifiedSamira.csv")
+
+features <- readRDS(paste("/data/srlab/jatin/cmqtl/gwas_data/final/donor_level/",args[1],".final.rds", sep=""))
+
+Covs <- c("FID",
+        "IID",
+        "Metadata_Plate",
+        "Metadata_Sex")
+
+qCovs <- c("FID",
+	"IID", 
+	"PC1",
+        "PC2",
+        "PC3",
+        "PC4",
+	"Cells_Neighbors_NumberOfNeighbors_Adjacent",
+	"Metadata_onEdge")
+
+ExcludeColumns <- c("Metadata_line_ID",
+	"Metadata_ID",
+	"Metadata_Plate",
+	"Metadata_Clinical_Diagnosis",
+	"Metadata_Age",
+	"Metadata_Sex",
+	"Metadata_iPSC_Origin",
+	"Metadata_Doubling_Hour",
+	"Metadata_Disease_Status" ,
+	"Metadata_Disease_Category",
+	"Metadata_CellCount_avg",
+	"PC1",
+	"PC2",
+	"PC3",
+	"PC4",
+	"PC5",
+	"Cells_Neighbors_NumberOfNeighbors_Adjacent",
+	"Metadata_onEdge")
+
+### for isolate i used the following lists instead
+#qCovs <- c("FID",
+#        "IID",
+#	"PC1",
+#        "PC2",
+#        "PC3",
+#        "PC4",
+#	"Metadata_onEdge")
+#
+#ExcludeColumns <- c("Metadata_line_ID",
+#        "Metadata_ID",
+#        "Metadata_Plate",
+#        "Metadata_Clinical_Diagnosis",
+#        "Metadata_Age",
+#        "Metadata_Sex",
+#        "Metadata_iPSC_Origin",
+#        "Metadata_Doubling_Hour",
+#        "Metadata_Disease_Status" ,
+#        "Metadata_Disease_Category",
+#	"Metadata_CellCount_avg",
+#        "PC1",
+#        "PC2",
+#        "PC3",
+#        "PC4",
+#	"PC5",
+#	"Metadata_onEdge")
+#
+## order data like fam
+data <- merge(id_map, features, by=c("Metadata_line_ID","Metadata_ID"))
+
+fam <- read.table("../SecondRound_JatinFeatures/PlinkFiles/V2F_chr1.nodup.fam", header=F)
+fam <- fam[,1:2]
+colnames(fam) <- c("FID", "IID")
+
+new_data <- merge(fam, data, by.x="FID", by.y="wgs_id", all.x=T)
+new_data <- new_data %>% group_by(Metadata_ID) %>% sample_n(1)
+new_data <- new_data[match(fam$IID, new_data$IID),]
+new_data <- subset(new_data, !is.na(Metadata_ID))
+
+### make pheno and cov files
+new_data <- as.data.frame(new_data)
+pheno <-  new_data %>% dplyr::select(-c(all_of(ExcludeColumns)))
+covar <- new_data %>% dplyr::select(c(all_of(Covs)))
+qcovar <- new_data %>% dplyr::select(c(all_of(qCovs)))
+
+write.table(colnames(pheno[,-c(1:2)]), paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/",args[1],"/",args[1],".phenotypeNames.txt", sep=""), quote=F, row.names=T, col.names=F)
+write.table(colnames(covar[,-c(1:2)]), paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/",args[1],"/",args[1],".CatCovarNames.txt", sep=""), quote=F, row.names=T, col.names=F)
+write.table(colnames(qcovar[,-c(1:2)]), paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/",args[1],"/",args[1],".QuanCovarNames.txt", sep=""), quote=F, row.names=T, col.names=F)
+
+write.table(pheno, paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/",args[1],"/",args[1],".pheno", sep=""), quote=F, row.names=F, col.names=F)
+write.table(qcovar, paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/",args[1],"/",args[1],".quantitative.cov", sep=""), quote=F, row.names=F, col.names=F)
+write.table(covar, paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/",args[1],"/",args[1],".categorical.cov", sep=""), quote=F, row.names=F, col.names=F)
+

02_RunAssociation.sh

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+#!/bin/bash
+
+module load plink/1.90b3
+module load gcta/1.91.1
+
+export LSB_JOB_REPORT_MAIL=N
+
+function write_Rscript()
+{
+	local outDir=/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov
+	local chr=$1
+	local pheno=$2
+	local cell=$3
+
+cat << EOF | bsub
+
+export LSB_JOB_REPORT_MAIL=N
+#BSUB -L /bin/bash
+#BSUB -o STDfiles/fastgwa_${chr}_${pheno}_${cell}.out
+#BSUB -e STDfiles/fastgwa_${chr}_${pheno}_${cell}.err
+#BSUB -J fastgwa
+#BSUB -M 8000
+#BSUB -q rerunnable
+#BSUB -R 'rusage[mem=8000]'
+#BSUB -R 'select[hname!=cn001]'
+#BSUB -R 'select[hname!=cn002]'
+#BSUB -R 'select[hname!=cn003]'
+#BSUB -R 'select[hname!=cn004]'
+#BSUB -R 'select[hname!=cn005]'
+
+/data/srlab/sasgari/Software/gcta_1.93.2beta/gcta64 --bfile  ../SecondRound_JatinFeatures/PlinkFiles/V2F_chr${chr}.nodup.qc \
+--pheno ${outDir}/${cell}/${cell}.pheno \
+--mpheno ${pheno} \
+--covar ${outDir}/${cell}/${cell}.categorical.cov \
+--qcovar ${outDir}/${cell}/${cell}.quantitative.cov \
+--remove ../SecondRound_JatinFeatures/PlinkFiles/ExcludeFromAssociation.ID \
+--maf 0.05 \
+--fastGWA-lr \
+--threads 10 \
+--out ${outDir}/${cell}/${cell}_chr${chr}_pheno${pheno}.LR
+
+gzip -f ${outDir}/${cell}/${cell}_chr${chr}_pheno${pheno}.LR.fastGWA
+
+EOF
+}
+
+
+cat Chr_Pheno_Cell.list | while read chr pheno cell 
+	do
+	write_Rscript $chr $pheno $cell
+	done

03_GetLambda_QQ.R

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+library(qqman)
+library(data.table)
+library(readr)
+
+args <- commandArgs(trailingOnly = TRUE)
+
+raw.files <- Sys.glob(file.path(paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/", args[2],"/",args[2],"_chr*_pheno",args[1],".LR.fastGWA.gz", sep="")))
+
+results <- suppressWarnings(rbindlist(lapply(raw.files, function(filename) {suppressMessages(readr::read_tsv(filename, na = c("NaN")))})))
+
+results=na.omit(results)
+pvalues=results$P
+
+### qchisq(0.5,1) is the median of a chi-squared distribution with one degree of freedom
+z <- as.numeric(results$BETA)/as.numeric(results$SE) 
+lambda2 = round(median(z^2) / qchisq(0.5,1), 4)
+
+write.table(t(c(args[1], lambda2)), paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/QQ/", args[2],"/",args[2],"_pheno",args[1],".QQ.txt", sep=""), quote=F, col.names=F, row.names=F)
+
+#png(file=paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/QQ/", args[2],"/",args[2],"_pheno",args[1],".QQ.png", sep=""), width=400, height=400)
+#qqman::qq(pvalues, cex = 0.5, cex.axis = 1,main = paste(args[2], "pheno", args[1], "\n lambda=", lambda2, sep=" "))
+#dev.off()

03_Run.sh

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+#!/bin/bash
+module load R/3.4.0
+
+function write_Rscript()
+{
+        local pheno=$1
+        local cell=$2
+	module load R/3.4.0
+
+cat << EOF | bsub
+
+export LSB_JOB_REPORT_MAIL=N
+#BSUB -L /bin/bash
+#BSUB -o STDfiles/QQ_${pheno}_${cell}.out
+#BSUB -e STDfiles/QQ_${pheno}_${cell}.err
+#BSUB -J QQ
+#BSUB -M 12000
+#BSUB -q rerunnable
+#BSUB -R 'rusage[mem=12000]'
+#BSUB -R 'select[hname!=cn001]'
+#BSUB -R 'select[hname!=cn002]'
+#BSUB -R 'select[hname!=cn003]'
+#BSUB -R 'select[hname!=cn004]'
+#BSUB -R 'select[hname!=cn005]'
+
+/apps/lib-osver/R/3.4.0/bin/Rscript 03_GetLambda_QQ.R $pheno $cell
+ 
+EOF
+}
+
+cat Pheno_Cell.list | awk '{if ($1 < 4) print $0}' | while read pheno cell 
+        do
+        write_Rscript $pheno $cell
+        done

04_GetdataForManhattanPlot.R

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+library(data.table)
+library(readr)
+library(tidyverse)
+
+args <- commandArgs(trailingOnly = TRUE)
+
+raw.files <- Sys.glob(file.path(paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/", args[2],"/",args[2],"_chr*_pheno",args[1],".LR.fastGWA.gz", sep="")))
+
+results1 <- suppressWarnings(rbindlist(lapply(raw.files, function(filename) {suppressMessages(readr::read_tsv(filename, na = c("NaN")))})))
+results1=na.omit(results1)
+results1$P <- as.numeric(results1$P)
+
+sig <- subset(results1, P < 5e-8)
+
+#results1.1 <- results1 %>% filter(P <= 1e-2)
+#results1.2 <- results1 %>% filter(P > 1e-2) %>% sample_frac(0.01)
+#
+#results <- rbind(results1.1, results1.2)
+
+write.table(sig, paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/", args[2],"/",args[2],"_pheno",args[1],".lowP4manhattan.tmp", sep=""), quote=F, col.names=T, row.names=F, sep="\t")
+
+#write.table(results, paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/", args[2],"/",args[2],"_pheno",args[1],".DownSampled.tmp", sep=""), quote=F, col.names=T, row.names=F, sep="\t")
+

04_Run.sh

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+#!/bin/bash
+module load R/3.4.0
+
+function write_Rscript()
+{
+        local pheno=$1
+        local cell=$2
+	module load R/3.4.0
+
+cat << EOF | bsub
+
+export LSB_JOB_REPORT_MAIL=N
+#BSUB -L /bin/bash
+#BSUB -o STDfiles/ForManh_${pheno}_${cell}.out
+#BSUB -e STDfiles/ForManh_${pheno}_${cell}.err
+#BSUB -J DownSamp
+#BSUB -M 8000
+#BSUB -q rerunnable
+#BSUB -R 'rusage[mem=6000]'
+#BSUB -R 'select[hname!=cn001]'
+#BSUB -R 'select[hname!=cn002]'
+#BSUB -R 'select[hname!=cn003]'
+#BSUB -R 'select[hname!=cn004]'
+#BSUB -R 'select[hname!=cn005]'
+
+/apps/lib-osver/R/3.4.0/bin/Rscript 04_GetdataForManhattanPlot.R $pheno $cell
+
+#gzip -f /data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/${cell}/${cell}_pheno${pheno}.DownSampled.tmp
+ 
+EOF
+}
+
+cat Pheno_Cell.list | while read pheno cell 
+        do
+        write_Rscript $pheno $cell
+        done
+

05_ManhattanPlot.R

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+library(qqman)
+library(tidyverse)
+library(data.table)
+library(readr)
+
+args <- commandArgs(trailingOnly = TRUE)
+
+base.files <- Sys.glob(file.path(paste("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/", args[1],"/",args[1],"*.DownSampled.tmp.gz", sep="")))
+
+results1 <- suppressWarnings(rbindlist(lapply(base.files, function(filename) {suppressMessages(readr::read_tsv(filename, na = c("NaN")))})))
+results1=na.omit(results1)
+
+results1.1 <- results1 %>% filter(P <= 1e-3)
+results1.2 <- results1 %>% filter(P > 1e-3) %>% sample_frac(0.05)
+
+results <- rbind(results1.1, results1.2)
+
+##fdr <- fread(paste("gzip -dc FDRsigPvalue_", args[1], ".txt.gz", sep=""))
+##x <- nrow(fdr)
+##if(x == 0){
+##sug <- -log10(5e-8/908)
+##} else {
+##sug <- -log10(max(fdr$V1))
+##}
+
+png(file=paste(args[1], "_AllPheno_downSampled.png", sep=""), width=900, height=400)
+manhattan(results, chr="CHR", bp="POS", snp="SNP", p="P", suggestiveline = -log10(5e-8), genomewideline = -log10(5e-8/246) ,col=c("coral","blueviolet"), ylim=c(0, -log10(min(results$P))+1))
+dev.off()

06_PapersTable.R

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+library(readr)
+library(stringr)
+library(dplyr)
+library(data.table)
+
+pheno <- read.table("/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/isolate/isolate.phenotypeNames.txt", header=F)
+colnames(pheno) <- c("Pheno", "Feature")
+pheno$Pheno <- paste0("pheno", pheno$Pheno)
+
+raw.files <- Sys.glob(file.path(
+  "/data/srlab2/sasgari/Projects/V2F/Analysis/GWAS/FourthdeRound_TwoGroupEdgeCov/*/*_pheno*.lowP4manhattan.tmp"
+))
+
+data <- suppressWarnings(rbindlist(lapply(raw.files, function(filename) {
+    suppressMessages(readr::read_tsv(filename, col_names=FALSE))}), idcol = "File"))
+data[, File := factor(File, labels = basename(raw.files))]
+data <- data %>% filter(X1 != "CHR")
+data$File <- str_remove(data$File, ".lowP4manhattan.tmp")
+
+colnames(data) <-  c("File", "CHR", "SNP", "POS", "A1", "A2", "N", "AF1", "BETA","SE","P")
+data$Cell <- str_split_fixed(data$File, "_", 2)[,1]
+data$Pheno <- str_split_fixed(data$File, "_", 2)[,2]
+
+data <- merge(data, pheno, by="Pheno")
+data$P <- as.numeric(data$P)
+data <- data %>% filter(P <= 4.1e-8)
+write.table(data, "SuggestiveGWAS_results.txt", quote=F, col.names=T, row.names=F, sep="\t")