Approach to better assign genes to peaks

New Approach Idea

Lin et al: An in-depth map of polyadenylation sites in cancer 2012: -Mapped locations to annotated locations in UCUS browser: “The mapped locations were annotated using the UCSC genome browser tables ( 26 ). When a locus could be attributed to multiple possible annotations, the locus was assigned with a single annotation in the following priority order: 3′ UTRs (sense), coding sequences (CDS, sense), 5′ UTRs (sense), intron (sense), non-coding RNAs (ncRNAs, sense), 5′ UTR antisense, CDS antisense, 3′ UTR antisense, intron antisense, promoter antisense, ncRNA antisense and intergenic”

I want to download this annotation and try this. I am using the ncbi_refseq annotations. I will download regions of the genome seperatly and then merge the files.

• 5’ UTR

• Coding Exon

• Intron

• 3’ UTR

• (downstream 5000)-downstream proximal region

I also want a dictionary with the transcripts and the gene names for the annotation. This information will come from the Transcript2GeneName file. In this file the transcript ID is in column1 and the gene name column 13.

I have downloaded all of the these to data/RefSeq_annotations. I will concatinate all of these for a full annotation dataset, I will then sort this file. The file is ncbiRefSeq_allAnnotation.sort.bed

Using this I can create an annotation in a bed file I can use for the overlap with my peaks. This will include getting the transcript to gene annotations. I will transfer the files to midway in my genome annotation directory and work with them there.

Format full refseq annotation:

TXN2Gene_file="/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/Transcript2GeneName.dms"

gene_dic={}

for ln in open(TXN2Gene_file,"r"):
   txn=ln.split()[1]
   gene=ln.split()[12]
   gene_dic[txn]=gene

outF=open("/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_FormatedallAnnotation.sort.bed","w")

inFile="/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_allAnnotation.sort.bed"  


for ln in open(inFile, "r"):
   chrom, start, end, name, score, strand = ln.split()
   chrom_fix=chrom[3:]
   txn=name.split("_")[:2]
   txnF="_".join(txn)
   gene=gene_dic[txnF]
   type=name.split("_")[2]
   id=type + ":" + gene
   outF.write("%s\t%s\t%s\t%s\t%s\t%s\n"%(chrom_fix, start, end, id, score, strand))

outF.close()

Map peaks with new annotation

I want to create a file with my peaks mapped to these regions. I will include a structure for when there is a tie and put intergenic if it is not found. I need to do an intersect that gives me all of the IDs. After this I can use python to parse the hiarchy.

I can use bedtools map for this. I want all of the data to come back.

-c 4 -o distinct
-S opposite strand

I will do this on the peaks before I looked at usage.

mapnoMPPeaks2GenomeLoc.sh

#!/bin/bash

#SBATCH --job-name=mapnoMPPeaks2GenomeLoc
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=mapnoMPPeaks2GenomeLoc.out
#SBATCH --error=mapnoMPPeaks2GenomeLoc.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

#annotation: /project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_FormatedallAnnotation.sort.bed

#peaks:  /project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_filtered/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR.bed


bedtools map -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_filtered/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR.bed -b /project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_FormatedallAnnotation.sort.bed -c 4 -S -o distinct > /project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLoc.bed

Look at how many get no annotation. I can do this interactively in python.

num=0
for ln in open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLoc.bed", "r"):
    location=ln.split()[7]
    if location==".":
        num +=1

print(num)

This shows me that 85% of the peaks fall into one of these annotations.

Now I need to sort out the peaks with multiple annotations.

4. 5’ UTR

5. Coding Exon

6. Intron

7. 3’ UTR

8. (downstream 5000)-downstream proximal region

I can write this out as the SAF I need. GeneID (peak1:1:14404:14484:-:OR4F16) Chr Start End Strand. For this I cannot include the peaks with no gene association. I can go back to this if i need to in the future.

processGenLocPeakAnno2SAF.py

inFile="/project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLoc.bed"
outFile=open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.SAF" , "w")

outFile.write("GeneID\tChr\tStart\tEnd\tStrand\n")
for ln in open(inFile, "r"):
    chrom, start, end, peak, cov, strand, score, anno = ln.split()
    if anno==".": 
        continue  
    anno_lst=anno.split(",")
    if len(anno_lst)==1:
        gene=anno_lst[0].split(":")[1]
        print("1 gene")
        peak_i=int(peak)
        start_i=int(start)
        end_i=int(end)
        ID="peak%d:%s:%d:%d:%s:%s"%(peak_i, chrom, start_i, end_i, strand, gene)
        outFile.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
    else:
        type_dic={}
        for each in anno_lst:
            type_dic[each.split(":")[0]]=each.split(":")[1]
        if "utr3" in type_dic.keys():
            gene=type_dic["utr3"]
            peak_i=int(peak)
            start_i=int(start)
            end_i=int(end)
            ID="peak%d:%s:%d:%d:%s:%s"%(peak_i, chrom, start_i, end_i, strand, gene)
            outFile.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
        elif "end" in type_dic.keys():
            gene=type_dic["end"]
            peak_i=int(peak)
            start_i=int(start)
            end_i=int(end)
            ID="peak%d:%s:%d:%d:%s:%s"%(peak_i, chrom, start_i, end_i, strand, gene)
            outFile.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
        elif "cds" in type_dic.keys():
            gene=type_dic["cds"] 
            peak_i=int(peak)
            start_i=int(start)
            end_i=int(end)
            ID="peak%d:%s:%d:%d:%s:%s"%(peak_i, chrom, start_i, end_i, strand, gene)
            outFile.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
        elif "utr5" in type_dic.keys():
            gene=type_dic["utr5"]
            peak_i=int(peak)
            start_i=int(start)
            end_i=int(end)
            ID="peak%d:%s:%d:%d:%s:%s"%(peak_i, chrom, start_i, end_i, strand, gene)
            outFile.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
        elif "intron" in type_dic.keys():
            gene=type_dic["intron"]
            peak_i=int(peak)
            start_i=int(start)
            end_i=int(end)
            ID="peak%d:%s:%d:%d:%s:%s"%(peak_i, chrom, start_i, end_i, strand, gene)
            outFile.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))

outFile.close()

Map reads to new annotated peaks

This has all of the peaks with at least one gene annotation. (104555) I can run feature counts on this to start getting the usage.

GeneLocAnno_fc_TN_noMP.sh

Because I mapped the genes opposite. The reads are now on in the same direction as the peaks

#!/bin/bash

#SBATCH --job-name=GeneLocAnno_fc_TN_noMP
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=GeneLocAnno_fc_TN_noMP.out
#SBATCH --error=GeneLocAnno_fc_TN_noMP.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fc /project2/gilad/briana/threeprimeseq/data/bam_NoMP_sort/*T-combined-sort.noMP.sort.bam -s 1

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fc /project2/gilad/briana/threeprimeseq/data/bam_NoMP_sort/*N-combined-sort.noMP.sort.bam -s 1

Around 4 mill mapping in nuclear and 7 mill in total.

fix_head_fc_geneLoc_tot_noMP.py

infile= open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

fix_head_fc_geneLoc_nuc_noMP.py

infile= open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

I can use the fileIDs

/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP/file_id_mapping_nuclear_Transcript.txt and /project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP/file_id_mapping_total_Transcript.txt

Make phenotypes

I need to make a file with the gene start and ends because I use these in the phenotypes for QTL mapping. I can do this with the project2/gilad/briana/genome_anotation_data/RefSeq_annotations/Transcript2GeneName.dms file. I want the file to look like chr,start,end,txn:gene,.,strand I will sue the start and end of the coding seq

getGeneEnds.py

TXN2Gene_file=open("/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/Transcript2GeneName.dms","r")

outFile=open("/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_endAllGenes.bed", "w")

for i, ln in enumerate(TXN2Gene_file):
    if i >0 :
        chrom=ln.split()[2]
        chromf=chrom[3:]
        start=int(ln.split()[6])
        end=int(ln.split()[7])
        txn=ln.split()[1]
        genename=ln.split()[12]
        id=txn + ":" + genename
        strand=ln.split()[3]
        score="."
        outFile.write("%s\t%s\t%s\t%s\t%s\t%s\n"%(chromf, start, end, id, score, strand))

outFile.close()

Sort the bed file:

sort -k1,1 -k2,2n /project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_endAllGenes.bed > /project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_endAllGenes.sort.bed

makePhenoRefSeqPeaks_GeneLoc_Total_noMP.py

#PYTHON 3

dic_IND = {}
dic_BAM = {}

for ln in open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP/file_id_mapping_total_Transcript.txt"):
    bam, IND = ln.split("\t")
    IND = IND.strip()
    dic_IND[bam] = IND
    if IND not in dic_BAM:
        dic_BAM[IND] = []
    dic_BAM[IND].append(bam)


#now I have ind dic with keys as the bam and ind as the values
#I also have a bam dic with ind as the keys and bam as the values  
    
inds=list(dic_BAM.keys()) #list of ind libraries  

#gene start and end dictionaries: 
dic_geneS = {}
dic_geneE = {}
for ln in open("/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_endAllGenes.sort.bed"):
    chrom, start, end, geneID, score, strand = ln.split('\t')
    gene= geneID.split(":")[1]
#    if "-" in gene:
 #       gene=gene.split("-")[0]
    if gene not in dic_geneS:
        dic_geneS[gene]=int(start)
        dic_geneE[gene]=int(end)
        


#list of genes   

count_file=open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.fc", "r")
genes=[]
for line , i in enumerate(count_file):
    if line > 1:
        i_list=i.split()
        id=i_list[0]
        id_list=id.split(":")
        gene=id_list[5]
        if gene not in genes:
            genes.append(gene)
            
#make the ind and gene dic  
dic_dub={}
for g in genes:
    dic_dub[g]={}
    for i in inds:
        dic_dub[g][i]=0


#populate the dictionary  
count_file=open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.fc", "r")
for line, i in enumerate(count_file):
    if line > 1:
        i_list=i.split()
        id=i_list[0]
        id_list=id.split(":")
        g= id_list[5]
        values=list(i_list[6:])
        list_list=[]
        for ind,val in zip(inds, values):
            list_list.append([ind, val])
        for num, name in enumerate(list_list):
            dic_dub[g][list_list[num][0]] += int(list_list[num][1])
        

#write the file by acessing the dictionary and putting values in the table ver the value in the dic 
        

fout=open("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno.fc","w")
peak=["chrom"]
inds_noL=[]
for each in inds:
    indsNA= "NA" + each[:-2]
    inds_noL.append(indsNA) 
fout.write(" ".join(peak + inds_noL) + '\n' )


count_file=open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.fc", "r")
for line , i in enumerate(count_file):
    if line > 1:
        i_list=i.split()
        id=i_list[0]
        id_list=id.split(":")
        gene=id_list[5]
        start=dic_geneS[id_list[5]]
        end=dic_geneE[id_list[5]]
        buff=[]
        buff.append("chr%s:%d:%d:%s_%s_%s"%(id_list[1], start, end, id_list[5], id_list[4], id_list[0]))
        for x,y in zip(i_list[6:], inds):
            b=int(dic_dub[gene][y])
            t=int(x)
            buff.append("%d/%d"%(t,b))
        fout.write(" ".join(buff)+ '\n')
        
fout.close()

makePhenoRefSeqPeaks_GeneLoc_Nuclear_noMP.py

#PYTHON 3

dic_IND = {}
dic_BAM = {}

for ln in open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP/file_id_mapping_nuclear_Transcript.txt"):
    bam, IND = ln.split("\t")
    IND = IND.strip()
    dic_IND[bam] = IND
    if IND not in dic_BAM:
        dic_BAM[IND] = []
    dic_BAM[IND].append(bam)


#now I have ind dic with keys as the bam and ind as the values
#I also have a bam dic with ind as the keys and bam as the values  
    
inds=list(dic_BAM.keys()) #list of ind libraries  

#gene start and end dictionaries: 
dic_geneS = {}
dic_geneE = {}
for ln in open("/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_endAllGenes.sort.bed"):
    chrom, start, end, geneID, score, strand = ln.split('\t')
    gene= geneID.split(":")[1]
    #if "-" in gene:
     #   gene=gene.split("-")[0]
    if gene not in dic_geneS:
        dic_geneS[gene]=int(start)
        dic_geneE[gene]=int(end)
        


#list of genes   

count_file=open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.fc", "r")
genes=[]
for line , i in enumerate(count_file):
    if line > 1:
        i_list=i.split()
        id=i_list[0]
        id_list=id.split(":")
        gene=id_list[5]
        if gene not in genes:
            genes.append(gene)
            
#make the ind and gene dic  
dic_dub={}
for g in genes:
    dic_dub[g]={}
    for i in inds:
        dic_dub[g][i]=0


#populate the dictionary  
count_file=open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.fc", "r")
for line, i in enumerate(count_file):
    if line > 1:
        i_list=i.split()
        id=i_list[0]
        id_list=id.split(":")
        g= id_list[5]
        values=list(i_list[6:])
        list_list=[]
        for ind,val in zip(inds, values):
            list_list.append([ind, val])
        for num, name in enumerate(list_list):
            dic_dub[g][list_list[num][0]] += int(list_list[num][1])
        

#write the file by acessing the dictionary and putting values in the table ver the value in the dic 
        

fout=open("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno.fc","w")
peak=["chrom"]
inds_noL=[]
for each in inds:
    indsNA= "NA" + each[:-2]
    inds_noL.append(indsNA) 
fout.write(" ".join(peak + inds_noL) + '\n' )


count_file=open("/project2/gilad/briana/threeprimeseq/data/filtPeakOppstrand_cov_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.fc", "r")
for line , i in enumerate(count_file):
    if line > 1:
        i_list=i.split()
        id=i_list[0]
        id_list=id.split(":")
        gene=id_list[5]
        start=dic_geneS[id_list[5]]
        end=dic_geneE[id_list[5]]
        buff=[]
        buff.append("chr%s:%d:%d:%s_%s_%s"%(id_list[1], start, end, id_list[5], id_list[4], id_list[0]))
        for x,y in zip(i_list[6:], inds):
            b=int(dic_dub[gene][y])
            t=int(x)
            buff.append("%d/%d"%(t,b))
        fout.write(" ".join(buff)+ '\n')
        
fout.close()

Run make pheno files:

run_makePhen_sep_GeneLocAnno_noMP.sh

#!/bin/bash

#SBATCH --job-name=run_makePhen_sep_GeneLocAnno_noMP
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=run_makePhen_sep_GeneLocAnno_noMP.out
#SBATCH --error=run_makePhen_sep_GeneLocAnno_noMP.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

python makePhenoRefSeqPeaks_GeneLoc_Total_noMP.py  

python makePhenoRefSeqPeaks_GeneLoc_Nuclear_noMP.py  

Convert to Usage

pheno2CountOnly_genelocAnno.R

library(reshape2)
library(tidyverse)


totalPeakUs=read.table("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno.fc", header = T, stringsAsFactors = F) %>% separate(chrom, sep = ":", into = c("chr", "start", "end", "id")) %>% separate(id, sep="_", into=c("gene", "strand", "peak"))
nuclearPeakUs=read.table("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno.fc", header = T, stringsAsFactors = F) %>% separate(chrom, sep = ":", into = c("chr", "start", "end", "id")) %>% separate(id, sep="_", into=c("gene", "strand", "peak"))



write.table(totalPeakUs[,7:dim(totalPeakUs)[2]], file="/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno.CountsOnly",quote=FALSE, col.names = F, row.names = F)

write.table(nuclearPeakUs[,7:dim(nuclearPeakUs)[2]], file="/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno.CountsOnly",quote=FALSE, col.names = F, row.names = F)

convertCount2Numeric_noMP_GeneLocAnno.py

def convert(infile, outfile):
  final=open(outfile, "w")
  for ln in open(infile, "r"):
    line_list=ln.split()
    new_list=[]
    for i in line_list:
      num, dem = i.split("/")
      if dem == "0":
        perc = "0.00"
      else:
        perc = int(num)/int(dem)
        perc=round(perc,2)
        perc= str(perc)
      new_list.append(perc)
    final.write("\t".join(new_list)+ '\n')
  final.close()
  
convert("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno.CountsOnly","/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno.CountsOnlyNumeric.txt")


convert("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno.CountsOnly","/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno.CountsOnlyNumeric.txt")

Pull these into R here so I can filter peaks with 5% and understand how many per gene.

library(workflowr)

This is workflowr version 1.1.1
Run ?workflowr for help getting started

library(tidyverse)

── Attaching packages ───────────────────────────────────────────────────────────────────────── tidyverse 1.2.1 ──

✔ ggplot2 3.0.0     ✔ purrr   0.2.5
✔ tibble  1.4.2     ✔ dplyr   0.7.6
✔ tidyr   0.8.1     ✔ stringr 1.3.1
✔ readr   1.1.1     ✔ forcats 0.3.0

── Conflicts ──────────────────────────────────────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()

library(reshape2)

Attaching package: 'reshape2'

The following object is masked from 'package:tidyr':

    smiths

library(cowplot)

Attaching package: 'cowplot'

The following object is masked from 'package:ggplot2':

    ggsave

library(ggpubr)

Loading required package: magrittr

Attaching package: 'magrittr'

The following object is masked from 'package:purrr':

    set_names

The following object is masked from 'package:tidyr':

    extract

Attaching package: 'ggpubr'

The following object is masked from 'package:cowplot':

    get_legend

totalPeakUs=read.table("../data/PeakUsage_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno.fc", header = T, stringsAsFactors = F) %>% separate(chrom, sep = ":", into = c("chr", "start", "end", "id")) %>% separate(id, sep="_", into=c("gene", "strand", "peak"))

Warning: Expected 3 pieces. Additional pieces discarded in 4 rows [13402,
13403, 13404, 101569].

nuclearPeakUs=read.table("../data/PeakUsage_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno.fc", header = T, stringsAsFactors = F) %>% separate(chrom, sep = ":", into = c("chr", "start", "end", "id")) %>% separate(id, sep="_", into=c("gene", "strand", "peak"))

Warning: Expected 3 pieces. Additional pieces discarded in 4 rows [13402,
13403, 13404, 101569].

ind=colnames(totalPeakUs)[7:dim(totalPeakUs)[2]]
totalPeakUs_CountNum=read.table("../data/PeakUsage_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno.CountsOnlyNumeric.txt", col.names = ind)

nuclearPeakUs_CountNum=read.table("../data/PeakUsage_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno.CountsOnlyNumeric.txt", col.names = ind)

#numeric with anno
totalPeak=as.data.frame(cbind(totalPeakUs[,1:6], totalPeakUs_CountNum))
nuclearPeak=as.data.frame(cbind(nuclearPeakUs[,1:6], nuclearPeakUs_CountNum))

#mean
totalPeakUs_CountNum_mean=rowMeans(totalPeakUs_CountNum)
nuclearPeakUs_CountNum_mean=rowMeans(nuclearPeakUs_CountNum)


#append mean to anno
TotalPeakUSMean=as.data.frame(cbind(totalPeakUs[,1:6],totalPeakUs_CountNum_mean))
NuclearPeakUSMean=as.data.frame(cbind(nuclearPeakUs[,1:6],nuclearPeakUs_CountNum_mean))

Filter on the mean

TotalPeakUSMean_filt=TotalPeakUSMean %>% filter(totalPeakUs_CountNum_mean>=.05) %>% group_by(gene) %>% summarise(Npeaks=n())
totalPeaksPerGene=TotalPeakUSMean_filt %>% group_by(Npeaks) %>% summarise(GenesWithNPeaks=n())


NuclearPeakUSMean_filt=NuclearPeakUSMean %>% filter(nuclearPeakUs_CountNum_mean>=.05) %>% group_by(gene) %>% summarise(Npeaks=n())
nuclearPeaksPerGene=NuclearPeakUSMean_filt %>% group_by(Npeaks) %>% summarise(GenesWithNPeaks=n())
nuclearPeaksPerGene$GenesWithNPeaks=as.integer(nuclearPeaksPerGene$GenesWithNPeaks)

Peak num level

nPeaksBoth=totalPeaksPerGene %>% full_join(nuclearPeaksPerGene, by="Npeaks")
colnames(nPeaksBoth)= c("Peaks", "Total", "Nuclear")
nPeaksBoth$Total= nPeaksBoth$Total %>% replace_na(0)

#melt nPeaksBoth
nPeaksBoth_melt=melt(nPeaksBoth, id.var="Peaks")
colnames(nPeaksBoth_melt)= c("Peaks", "Fraction", "Genes")

#plot
peakUsage5perc=ggplot(nPeaksBoth_melt, aes(x=Peaks, y=Genes, fill=Fraction)) + geom_bar(stat="identity", position = "dodge") + labs(title="Number of Genes with >5% Peak Usage \n cleaned for mispriming") + theme(axis.text.y = element_text(size=12),axis.title.y=element_text(size=10,face="bold"), axis.title.x=element_text(size=12,face="bold"))+ scale_fill_manual(values=c("darkviolet","deepskyblue3"))  + facet_grid(~Fraction)

peakUsage5perc

This is a similar distribution to the other annotations.

Save this plot:

ggsave(peakUsage5perc, file="../output/plots/QC_plots/peakUsage5perc_noMP_geneLocAnno.png")

Saving 7 x 5 in image

Genes covered with these annoations.

#nuclear
nrow(NuclearPeakUSMean_filt) 

[1] 14851

#total
nrow(TotalPeakUSMean_filt) 

[1] 14852

There are more genes in this. We expect this because i have included LINCs.

Look at number of peaks in each set.

#nuclear  
NuclearPeakUSMean %>% filter(nuclearPeakUs_CountNum_mean>=.05) %>% nrow()

[1] 37370

#total
TotalPeakUSMean %>% filter(totalPeakUs_CountNum_mean>=.05) %>% nrow()

[1] 33002

There are a few less peaks. This is expected give we cut 15% of the peaks because they are not within 5kb of an annotated gene.

Write out these peaks

NuclearPeakUSMean_5perc=NuclearPeakUSMean %>% filter(nuclearPeakUs_CountNum_mean>=.05)
write.table(NuclearPeakUSMean_5perc,file="../data/PeakUsage_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno.NoMP_sm_quant.Nuclear_fixed.pheno.5percPeaks.txt", row.names=F, col.names=F, quote = F)


TotalPeakUSMean_5per= TotalPeakUSMean %>% filter(totalPeakUs_CountNum_mean>=.05) 
write.table(TotalPeakUSMean_5per,file="../data/PeakUsage_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno.NoMP_sm_quant.Total_fixed.pheno.5percPeaks.txt", row.names=F, col.names=F, quote = F)

I want to filter these peaks so I can rerun the leafcutter analysis and look at some of the results. I transfered these to /project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/.

filterPheno_bothFraction_GeneLocAnno_5perc.py

#python  

totalokPeaks5perc_file="/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno.NoMP_sm_quant.Total_fixed.pheno.5percPeaks.txt"

totalokPeaks5perc={}
for ln in open(totalokPeaks5perc_file,"r"):
    peakname=ln.split()[5]
    totalokPeaks5perc[peakname]=""


nuclearokPeaks5perc_file="/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno.NoMP_sm_quant.Nuclear_fixed.pheno.5percPeaks.txt"
nuclearokPeaks5perc={}
for ln in open(nuclearokPeaks5perc_file,"r"):
    peakname=ln.split()[5]
    nuclearokPeaks5perc[peakname]=""
    
    
totalPhenoBefore=open("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno.fc","r")
totalPhenoAfter=open("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno_5percUs/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Total.fixed.pheno_5perc.fc", "w")
for num, ln in enumerate(totalPhenoBefore):
    if num ==0:
        totalPhenoAfter.write(ln)
    else:  
        id=ln.split()[0].split(":")[3].split("_")[2]
        if id in totalokPeaks5perc.keys():
            totalPhenoAfter.write(ln)
totalPhenoAfter.close()  

nuclearPhenoBefore=open("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno.fc","r")
nuclearPhenoAfter=open("/project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno_5percUs/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno_NoMP_sm_quant.Nuclear.fixed.pheno_5perc.fc", "w")
for num, ln in enumerate(nuclearPhenoBefore):
    if num ==0:
        nuclearPhenoAfter.write(ln)
    else:  
        id=ln.split()[0].split(":")[3].split("_")[2]
        if id in nuclearokPeaks5perc.keys():
            nuclearPhenoAfter.write(ln)
nuclearPhenoAfter.close() 

Diff Iso in leafcutter

Now I can use these peaks to get counts for leafcutter.

Session information

sessionInfo()

R version 3.5.1 (2018-07-02)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS  10.14.1

Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] bindrcpp_0.2.2  ggpubr_0.1.8    magrittr_1.5    cowplot_0.9.3  
 [5] reshape2_1.4.3  forcats_0.3.0   stringr_1.3.1   dplyr_0.7.6    
 [9] purrr_0.2.5     readr_1.1.1     tidyr_0.8.1     tibble_1.4.2   
[13] ggplot2_3.0.0   tidyverse_1.2.1 workflowr_1.1.1

loaded via a namespace (and not attached):
 [1] tidyselect_0.2.4  haven_1.1.2       lattice_0.20-35  
 [4] colorspace_1.3-2  htmltools_0.3.6   yaml_2.2.0       
 [7] rlang_0.2.2       R.oo_1.22.0       pillar_1.3.0     
[10] glue_1.3.0        withr_2.1.2       R.utils_2.7.0    
[13] modelr_0.1.2      readxl_1.1.0      bindr_0.1.1      
[16] plyr_1.8.4        munsell_0.5.0     gtable_0.2.0     
[19] cellranger_1.1.0  rvest_0.3.2       R.methodsS3_1.7.1
[22] evaluate_0.11     labeling_0.3      knitr_1.20       
[25] broom_0.5.0       Rcpp_0.12.19      scales_1.0.0     
[28] backports_1.1.2   jsonlite_1.5      hms_0.4.2        
[31] digest_0.6.17     stringi_1.2.4     grid_3.5.1       
[34] rprojroot_1.3-2   cli_1.0.1         tools_3.5.1      
[37] lazyeval_0.2.1    crayon_1.3.4      whisker_0.3-2    
[40] pkgconfig_2.0.2   xml2_1.2.0        lubridate_1.7.4  
[43] assertthat_0.2.0  rmarkdown_1.10    httr_1.3.1       
[46] rstudioapi_0.8    R6_2.3.0          nlme_3.1-137     
[49] git2r_0.23.0      compiler_3.5.1