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process_cutandrun_pausingindex.py

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+import os
+import numpy as np
+import pandas as pd
+import deeptools.countReadsPerBin as crpb
+import matplotlib
+import matplotlib.pyplot as plt
+import pysam
+import scipy.stats
+from statsmodels.stats.multitest import multipletests
+from pathlib import Path
+
+
+
+working_dir = "/omics/groups/OE0219/internal/Etienne/Projects/SRSF2/cutandrun/analysis/2022-11-09"
+Path(working_dir).mkdir(parents=True, exist_ok=True)
+windowsize_TSS = 500
+chromosomes = ["chr"+str(x) for x in range(1,23)] + ["chrX","chrY"]
+
+# Select downregulated genes (or upregulated, or background)
+chromosomes = [str(x) for x in range(1,23)] + ["X","Y"]
+#df = pd.read_csv("/omics/groups/OE0219/internal/Etienne/Projects/SRSF2/cutandrun/analysis/final_48kd_ctr_annotation.txt",sep="\t")
+df = pd.read_csv("/omics/groups/OE0219/internal/Etienne/Projects/SRSF2/cutandrun/analysis/res_TcRC_60min_kd_ctr.txt",sep=",")
+df.columns = ["gene_name","baseMean","log2FoldChange","lfcSE","stat","pvalue","padj"]
+df = df.loc[~df["pvalue"].isnull(),]
+df = df.loc[df["baseMean"]>5,:]
+df["padj"] = multipletests(df["pvalue"],alpha=0.05,method="fdr_bh")[1]
+
+df_downregulated = df.loc[ (df["padj"]<0.05) & (df["log2FoldChange"]<0),:] 
+genes_selected_downregulated = list(df_downregulated["gene_name"])
+
+df_upregulated = df.loc[ (df["padj"]<0.05) & (df["log2FoldChange"]>0),:]
+genes_selected_upregulated = list(df_upregulated["gene_name"])
+
+df_background = df.loc[ (df["baseMean"]>5),:] # (df["padj"]>0.20) & 
+genes_selected_background = list(df_background["gene_name"])
+
+# Find canonical transcripts for the selected genes
+#transcripts_ids = set()
+canonicalTranscripts2gene_downregulated = {}
+canonicalTranscripts2gene_upregulated = {}
+canonicalTranscripts2gene_background = {}
+df_GRCh38 = pd.read_csv("/omics/groups/OE0219/internal/Etienne/data/reference/RNA/Transcripts_GRCh38.tsv",sep="\t")
+for i in range(df_GRCh38.shape[0]):
+    if df_GRCh38.loc[i,"Ensembl Canonical"] == 1.0:
+        if df_GRCh38.loc[i,"Gene name"] in genes_selected_downregulated and not df_GRCh38.loc[i,"Gene name"] in canonicalTranscripts2gene_downregulated.values():
+            canonicalTranscripts2gene_downregulated[df_GRCh38.loc[i,"Transcript stable ID"]] = df_GRCh38.loc[i,"Gene name"]
+        if df_GRCh38.loc[i,"Gene name"] in genes_selected_upregulated and not df_GRCh38.loc[i,"Gene name"] in canonicalTranscripts2gene_upregulated.values():
+            canonicalTranscripts2gene_upregulated[df_GRCh38.loc[i,"Transcript stable ID"]] = df_GRCh38.loc[i,"Gene name"]
+        if df_GRCh38.loc[i,"Gene name"] in genes_selected_background and not df_GRCh38.loc[i,"Gene name"] in canonicalTranscripts2gene_background.values():
+            canonicalTranscripts2gene_background[df_GRCh38.loc[i,"Transcript stable ID"]] = df_GRCh38.loc[i,"Gene name"]
+
+df_GRCh37 = pd.read_csv("/omics/groups/OE0219/internal/Etienne/data/reference/RNA/Transcripts_GRCh37.tsv",sep="\t")
+df_GRCh37.sort_values(by=["Chromosome/scaffold name","Transcript start (bp)"],inplace=True)
+df_GRCh37.reset_index(inplace=True)
+
+with open(os.path.join(working_dir,"selected_genes_downregulated.bed"),"w") as outfile:
+    for i in range(df_GRCh37.shape[0]):
+        if df_GRCh37.loc[i,"Transcript stable ID"] in canonicalTranscripts2gene_downregulated and df_GRCh37.loc[i,"Chromosome/scaffold name"] in chromosomes:
+            strand = "+" if df_GRCh37.loc[i,"Strand"] >0 else "-"
+            tmp = outfile.write("\t".join(["chr"+str(df_GRCh37.loc[i,"Chromosome/scaffold name"]),str(df_GRCh37.loc[i,"Transcript start (bp)"]),str(df_GRCh37.loc[i,"Transcript end (bp)"]),canonicalTranscripts2gene_downregulated[df_GRCh37.loc[i,"Transcript stable ID"]],"0",strand])+"\n")
+
+with open(os.path.join(working_dir,"selected_genes_upregulated.bed"),"w") as outfile:
+    for i in range(df_GRCh37.shape[0]):
+        if df_GRCh37.loc[i,"Transcript stable ID"] in canonicalTranscripts2gene_upregulated and df_GRCh37.loc[i,"Chromosome/scaffold name"] in chromosomes:
+            strand = "+" if df_GRCh37.loc[i,"Strand"] >0 else "-"
+            tmp = outfile.write("\t".join(["chr"+str(df_GRCh37.loc[i,"Chromosome/scaffold name"]),str(df_GRCh37.loc[i,"Transcript start (bp)"]),str(df_GRCh37.loc[i,"Transcript end (bp)"]),canonicalTranscripts2gene_upregulated[df_GRCh37.loc[i,"Transcript stable ID"]],"0",strand])+"\n")
+
+with open(os.path.join(working_dir,"selected_genes_background.bed"),"w") as outfile:
+    for i in range(df_GRCh37.shape[0]):
+        if df_GRCh37.loc[i,"Transcript stable ID"] in canonicalTranscripts2gene_background and df_GRCh37.loc[i,"Chromosome/scaffold name"] in chromosomes:
+            strand = "+" if df_GRCh37.loc[i,"Strand"] >0 else "-"
+            tmp = outfile.write("\t".join(["chr"+str(df_GRCh37.loc[i,"Chromosome/scaffold name"]),str(df_GRCh37.loc[i,"Transcript start (bp)"]),str(df_GRCh37.loc[i,"Transcript end (bp)"]),canonicalTranscripts2gene_background[df_GRCh37.loc[i,"Transcript stable ID"]],"0",strand])+"\n")
+
+
+
+
+# Get dataframe of pausing indices transcripts x sample
+antibody = "phospho-Rpb1-ser5"
+replicates = ["1","2","3"]
+pipeline_dir = "/omics/groups/OE0219/internal/Etienne/Projects/SRSF2/cutandrun/pipeline2_RPKM/"
+
+#antibody = "RPB1"
+#replicates = ["3","4","5"]
+#pipeline_dir = "/omics/groups/OE0219/internal/Etienne/Projects/SRSF2/cutandrun/pipeline_RPKM/"
+
+sample2bam={}
+sample2pysam={}
+sample2counter={}
+for condition in ["KD48h","control"]:
+    for replicate in replicates:
+        sample = antibody+"_"+condition+"_R"+replicate
+        sample2bam[sample] = pipeline_dir+"02_alignment/bowtie2/target/"+condition+"-"+antibody+"_R"+replicate+".target.markdup.bam"
+        sample2pysam[sample] = pysam.AlignmentFile(sample2bam[sample])
+        sample2counter[sample] = crpb.CountReadsPerBin([sample2bam[sample]], binLength=100, stepSize=100)
+
+chromosomes = ["chr"+str(x) for x in range(1,23)] + ["chrX","chrY"]
+
+def compute_pausing_indices(genes_file,outfile):
+    # We define the pausing index as the number of reads aligned to the TSS (+/- 250bp) divided by the number of reads aligned to the gene body. 
+    d={"gene":[]}
+    for sample in sample2bam:
+        d[sample] = []
+    with open(genes_file,"r") as infile:
+        for line in infile:
+            linesplit= line.rstrip("\n").split("\t")
+            chr = linesplit[0]
+            if not chr in chromosomes: continue
+            start = int(linesplit[1])
+            end = int(linesplit[2])
+            gene = linesplit[3]
+            strand = linesplit[5]
+            if strand=="+":
+                TSS = start
+            else:
+                TSS = end
+            d["gene"].append(gene)
+            for condition in ["KD48h","control"]:
+                for replicate in replicates:
+                    sample =  antibody+"_"+condition+"_R"+replicate
+                    coverage_TSS = 1+sample2counter[sample].get_coverage_of_region(sample2pysam[sample],chr, [(TSS-windowsize_TSS//2, TSS+windowsize_TSS//2)])[0]
+                    coverage_genebody = 1+sample2counter[sample].get_coverage_of_region(sample2pysam[sample],chr, [(start -windowsize_TSS//2 , end +windowsize_TSS//2)])[0]
+                    ratio = coverage_TSS / coverage_genebody
+                    d[sample].append(ratio)
+    df_pausing_indices = pd.DataFrame(d)
+    df_pausing_indices.to_csv(outfile,sep="\t",index=False)
+
+compute_pausing_indices(os.path.join(working_dir,"selected_genes_downregulated.bed"),os.path.join(working_dir,antibody+"_pausing-indices_downregulated.tsv"))
+compute_pausing_indices(os.path.join(working_dir,"selected_genes_upregulated.bed"),os.path.join(working_dir,antibody+"_pausing-indices_upregulated.tsv"))
+compute_pausing_indices(os.path.join(working_dir,"selected_genes_background.bed"),os.path.join(working_dir,antibody+"_pausing-indices_background.tsv"))
+
+
+
+#######################################################
+# Compute pvalues for differences in pausing index between the two conditions
+
+for type in ["downregulated","upregulated","background"]:
+    print(type)
+    df_pausing_indices = pd.read_csv(os.path.join(working_dir,antibody+"_pausing-indices_"+type+".tsv"),sep="\t",index_col = "gene")
+    conditions = ["KD48h","control"]
+
+
+    # For each gene, perform a t-test between the pausing index of 2 conditions (requires several replicates per condition).
+    # Then, combine the pvalues of all the genes into a single pvalue
+
+    # Alternatively, for each gene, compute the log ratio of the pausing index between the two conditions, and then perform a t-test for all genes
+    pvalues = []
+    log_ratios_conditions=[]
+    diff_ratios_conditions=[]
+
+    for gene in df_pausing_indices.index:
+        # Collect the pausing indices for this gene, for each of the 2 conditions
+        pausing_indices={}
+        for condition in conditions:
+            pausing_indices[condition]=[]
+            for replicate in replicates: 
+                pausing_indices[condition].append(df_pausing_indices.loc[gene,antibody+"_"+condition+"_R"+str(replicate)])
+        # t-test between the two conditions
+        pvalue = scipy.stats.ttest_ind(pausing_indices[conditions[0]],pausing_indices[conditions[1]])[1] # ,alternative="greater"
+        if pvalue == pvalue:
+            pvalues.append(pvalue)
+
+        # log ratio between the 2 conditions
+        log_ratios_conditions.append(np.log(np.mean(pausing_indices[conditions[0]]) / np.mean(pausing_indices[conditions[1]])))
+        diff_ratios_conditions.append(np.mean(pausing_indices[conditions[0]]) - np.mean(pausing_indices[conditions[1]]))
+
+    ratios_condition = {}
+    for condition in conditions:
+        ratios_condition[condition] = []
+        for replicate in replicates:
+            ratios_replicate = []
+            for gene in df_pausing_indices.index:
+                ratios_replicate.append(df_pausing_indices.loc[gene,antibody+"_"+condition+"_R"+str(replicate)])
+            ratios_condition[condition].append(np.mean(ratios_replicate))
+
+    pvalue_samples = scipy.stats.ttest_ind(ratios_condition[conditions[0]],ratios_condition[conditions[1]])[1]
+    print(ratios_condition)
+    print("p value:" +str(pvalue_samples))
+
+    # use Fisher's method to combine the pvalues.
+    print(scipy.stats.combine_pvalues(pvalues))
+    pvalue_combined = scipy.stats.combine_pvalues(pvalues)[1]
+    print("pvalue combined: "+str(pvalue_combined))
+
+
+    # t-test between the two conditions
+    pvalue2 = scipy.stats.ttest_1samp(diff_ratios_conditions,0)[1] # alternative="greater"
+    print("pvalue diff: "+str(pvalue2))
+    pvalue3 = scipy.stats.ttest_1samp(log_ratios_conditions,0)[1] # alternative="greater"
+    print("pvalue log ratio: "+str(pvalue3))
+
+    matplotlib.rcParams.update({'font.size': 20})
+    #plt.hist(log_ratios_conditions,bins=np.arange(-1.6,1.6,0.1),density=False)
+    plt.hist(diff_ratios_conditions,bins=np.arange(-0.2,0.2,0.01),density=False)
+    plt.xlabel("Difference of pausing index between KD and control")
+    plt.ylabel("Number of transcripts")
+    plt.title("pvalue: "+ "{:.2E}".format(pvalue2))
+    plt.savefig(os.path.join(working_dir,antibody+"_ttest_"+type+".svg"),bbox_inches="tight")
+    plt.cla() 
+    plt.clf() 
+    plt.close('all')
+    series_diff_ratio = pd.Series(diff_ratios_conditions,index=df_pausing_indices.index)
+    series_diff_ratio.to_csv(os.path.join(working_dir,antibody+"_diff_pausing-indices_"+type+".tsv"),sep="\t")
+
+    matplotlib.rcParams.update({'font.size': 20})
+    #plt.hist(log_ratios_conditions,bins=np.arange(-1.6,1.6,0.1),density=False)
+    plt.hist(log_ratios_conditions,bins=np.arange(-0.2,0.2,0.01),density=False)
+    plt.xlabel("Log ratio of pausing index between KD and control")
+    plt.ylabel("Number of transcripts")
+    plt.title("pvalue: "+ "{:.2E}".format(pvalue3))
+    plt.savefig(os.path.join(working_dir,antibody+"_ttestLogRatio_"+type+".svg"),bbox_inches="tight")
+    plt.cla() 
+    plt.clf() 
+    plt.close('all')
+
+
+    plt.hist(pvalues)
+    plt.xlabel("pvalue")
+    plt.ylabel("Number of transcripts")
+    plt.title("Combined pvalue:" + "{:.2E}".format(pvalue_combined))
+    plt.savefig(os.path.join(working_dir,antibody+"_pvalues_"+type+".svg"),bbox_inches="tight")
+    plt.cla() 
+    plt.clf() 
+    plt.close('all')