peakQCplotsSTARprocessing.Rmd

---
title: "Peak Plots"
author: "Briana Mittleman"
date: "2/16/2019"
output: html_document
---

```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = TRUE)
```


```{r}
library(workflowr)
library(tidyverse)
library(reshape2)
library(cowplot)
```

I want to remake a lot of the peak QC plots I have been making with the new mapped and proccessed data created in the [accounting for mappping bias analysis](accountMapBias.html) 


* Peaks per gene  

* Number of genes with 1 peak, 2 peaks, more peaks  

* Distance between gene and TES  

* Peaks in each category  

* Peak Size 



###Peak per gene:  

I will do this for total and nuclear 5% seperatly then for the peaks I used in the QTL analysis.  

Nuclear peaks: 42127: /project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno.NoMP_sm_quant.Nuclear_fixed.pheno.5percPeaks.txt

Total peaks: 36915: /project2/gilad/briana/threeprimeseq/data/phenotypes_filtPeakTranscript_noMP_GeneLocAnno/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno.NoMP_sm_quant.Total_fixed.pheno.5percPeaks.txt


```{r}
peakNames=c("chr", 'start','end','gene','strand','name', 'mean')
totalPeaks=read.table("../data/PeaksUsed_noMP_5percCov/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno.NoMP_sm_quant.Total_fixed.pheno.5percPeaks.txt", stringsAsFactors = F, col.names = peakNames)
nuclearPeaks=read.table("../data/PeaksUsed_noMP_5percCov/filtered_APApeaks_merged_allchrom_refseqGenes.GeneLocAnno.NoMP_sm_quant.Nuclear_fixed.pheno.5percPeaks.txt", stringsAsFactors = F, col.names = peakNames)
```


Peaks per gene:  

```{r}
totalPeaks_genes=totalPeaks %>% group_by(gene) %>% summarise(nPeaks=n()) %>% group_by(nPeaks) %>% summarise(GenesWithNPeaks=n())
nuclearPeaks_genes=nuclearPeaks %>% group_by(gene) %>% summarise(nPeaks=n())%>% group_by(nPeaks) %>% summarise(GenesWithNPeaks=n())

nPeaksBoth=totalPeaks_genes %>% full_join(nuclearPeaks_genes, by="nPeaks")

colnames(nPeaksBoth)= c("Npeaks", "Total", "Nuclear")
nPeaksBoth$Total= nPeaksBoth$Total %>% replace_na(0)

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

peakUsage5perc=ggplot(nPeaksBoth_melt, aes(x=PAS, y=Genes, fill=Fraction)) + geom_bar(stat="identity", position = "dodge") + labs(title="Number of Genes by PAS Number \n 5% Usage",x="Number of PAS in Gene") + 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

ggsave(peakUsage5perc, file="../output/plots/PeakNumberPerGenebyFrac.png")
```

Plot this with the peaks used in the fraction

```{r}
allPeaks=read.table("../data/PeaksUsed_noMP_5percCov/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.5percCov.bed", stringsAsFactors = F, col.names = c("chr", 'start','end', 'id', 'score', 'strand')) %>% separate(id, into=c("gene", "peak"), sep=":")%>% group_by(gene) %>% summarise(nPeaks=n()) %>% group_by(nPeaks) %>% summarise(GenesWithNPeaks=n())

colnames(allPeaks)=c("PAS","Genes" )

allPeaksGenes=ggplot(allPeaks, aes(x=PAS, y=Genes)) + geom_bar(stat="identity",fill="blue") + labs(title="Number of Genes by PAS Count: \n PAS Used in QTL analysis",x="Number of PAS in Gene") + 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")) 

allPeaksGenes

ggsave(allPeaksGenes, file="../output/plots/PeakNumberPerGeneUsedinQTL.png")
```
### Number of genes with 1 peak, 2 peaks, more peaks  


Make this as a boxplot  

```{r}
GeneAnno=read.table("../data/RefSeq_annotations/Transcript2GeneName.dms", stringsAsFactors = F, header=T) %>% select(name2) %>%  unique()
colnames(GeneAnno)="gene"
genesWithpeak= read.table("../data/PeaksUsed_noMP_5percCov/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.5percCov.bed", stringsAsFactors = F, col.names = c("chr", 'start','end', 'id', 'score', 'strand')) %>% separate(id, into=c("gene", "peak"), sep=":") %>% select(gene) %>% unique()

Geneswith0= GeneAnno %>% anti_join(genesWithpeak, by="gene") %>% nrow()
Geneswith0
```


To get the genes with 0 peaks I need to pull in the gene annotation file  
```{r}

morethan2= allPeaks %>% filter(PAS > 2) 
colSums(morethan2)
Category=c("0 PAS", "1 PAS", "2 PAS", "More than 2 PAS")
genesPerCat=c(11896/27115, 4909/27115, 2949/27115, 7361/27115)

genesPerCat_df=as.data.frame(cbind(Category,genesPerCat))

genesPerCat_df$genesPerCat=as.numeric(as.character(genesPerCat_df$genesPerCat))

lab0=paste("Genes =", "11896", sep=" ")
lab1=paste("Genes =", "4909", sep=" ")
lab2=paste("Genes =", "2949", sep=" ")
labMore=paste("Genes =", "7361", sep=" ")


propGenesbyPAS=ggplot(genesPerCat_df, aes(x="", y=genesPerCat, fill=Category)) + geom_bar(stat="identity") + labs(x="Total Genes = 27115", y="Proportion of Genes", title="Proportion of Genes by number of PAS") + annotate("text", x="", y= .7, label=lab0) + annotate("text", x="", y= .5, label=lab1) + annotate("text", x="", y= .33, label=lab2) + annotate("text", x="", y= .2, label=labMore)

propGenesbyPAS


ggsave(propGenesbyPAS, file="../output/plots/PropOfGenesByPASnum.png")
```


###Distance between TES and peak  

* GetDistTXNend2Peak.py

convert /project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.5percCov.SAF to bed file

peaksGeneLocAnno_5percSAF2Bed.py
```{r}
distTXN2Peak=read.table("../data/DistTXN2Peak_genelocAnno/distPeak2EndTXN.txt", col.names = c("Peak", "name2", "Distance", "Gene_Strand"),stringsAsFactors = F)
txnanno=read.table("../data/RefSeq_annotations/Transcript2GeneName.dms", header=T,stringsAsFactors = F) %>% mutate(length=abs(txEnd-txStart)) %>% semi_join(distTXN2Peak, by="name2")
distTXN2Peak =distTXN2Peak %>% mutate(AbsDist=abs(Distance))
mean(txnanno$length)
```

```{r}
distTXN2PeakPlot=ggplot(distTXN2Peak, aes(x=AbsDist + 1)) + geom_density() + scale_x_log10() + labs(x="Absolute Distance between end of Transcription and center of Peak", title="Distribution of transcription to peak absolute distance") +  geom_vline(xintercept=mean(txnanno$length), col="red") + annotate("text", x=1000000, y=.4, label="Average transcript length \n for genes in peaks", col='red')

distTXN2PeakPlot

ggsave(distTXN2PeakPlot, file="../output/plots/DistanceBetweenPeakandTES.png")
```


###Peaks per category  

* processGenLocPeakAnno2SAF_withAnno.py
* filternamePeaks5percCov_GeneLocAnno_withAnno.py  

```{r}
peakswAnno=read.table("../data/PeaksUsed_noMP_5percCov/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.5percCov_withAnno.SAF", header=T) %>% separate(GeneID, into=c("Peak", "chrom", "start", "end", "strand", "gene", "loc"),sep=":") %>% select(Peak, loc) %>% group_by(loc) %>% summarise(Num=n())

```



```{r}
locationOfPeaks=ggplot(peakswAnno, aes(x=loc, y=Num)) + geom_bar(stat="identity", fill="blue") + labs(x="Gene Location", y="Number of Peaks", title="Location distribution for all PAS with 5% Usage")
locationOfPeaks
ggsave(locationOfPeaks, file="../output/plots/PeakLocationByAnnotation.png")
```


###Peak Size  

Peak length:  


```{r}
peaks=read.table("../data/PeaksUsed_noMP_5percCov/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.5percCov.bed",col.names=c("chr", 'start','end', 'peak', 'score', 'strand')) %>% mutate(length=end-start)

```

```{r}
ggplot(peaks,aes(x=length)) + geom_histogram(bins=300) + labs(title="Peak Size", x="number of basepairs") + geom_vline(xintercept =mean(peaks$length),col="red")
```


###Deep tools:  

files to remake:  

Merged bam files are in /project2/gilad/briana/threeprimeseq/data/mergedBams_NoMP

Code is mergeBam2BW.sh


mergeBam2BW.sh  

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=mergeBam2B
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=mergeBam2BW.out
#SBATCH --error=mergeBam2BW.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

#total  
bamCoverage -b /project2/gilad/briana/threeprimeseq/data/mergedBams_NoMP/AllTotalSamples.MergedBamFiles.noMP.sort.bam -o /project2/gilad/briana/threeprimeseq/data/mergedBW/Total_MergedBamCoverage.bw  

#nuclear  
bamCoverage -b /project2/gilad/briana/threeprimeseq/data/mergedBams_NoMP/AllNuclearSamples.MergedBamFiles.noMP.sort.bam -o /project2/gilad/briana/threeprimeseq/data/mergedBW/Nuclear_MergedBamCoverage.bw    
```

I need to remake the peak files with the strand opposite (peaks are opposite strand from the reads!)  


fixStrand4DTplots.py

```{bash,eval=F}
peaksIn="/project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.5percCov.bed"
intronIn="/project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed_5percCov_INTRON.bed"
PeakOut="/project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand.bed"
intronOut="/project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand_INTRON.bed"


def fix_strand(Fin,Fout):
    fout=open(Fout,"w")
    for ln in open(Fin, "r"):
        chrom, start, end, name, score, strand = ln.split()
        if strand=="+":
            fout.write("%s\t%s\t%s\t%s\t%s\t-\n"%(chrom,start,end,name,score))
        else:
            fout.write("%s\t%s\t%s\t%s\t%s\t+\n"%(chrom,start,end,name,score))
    fout.close()
    
    
fix_strand(peaksIn, PeakOut)
fix_strand(intronIn, intronOut)
```


/project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.5percCov.bed 

/project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed_5percCov_INTRON.bed



BothFracDTPlotmyPeaks_noMPFilt.sh

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=BothFracDTPlotmyPeaks_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=BothFracDTPlotmyPeaks_noMPFilt.out
#SBATCH --error=BothFracDTPlotmyPeaks_noMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/mergedBW/Total_MergedBamCoverage.bw /project2/gilad/briana/threeprimeseq/data/mergedBW/Nuclear_MergedBamCoverage.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand.bed -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFrac_myPeaksNompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFrac_myPeaksNompfilt.gz --refPointLabel "Called PAS" --plotTitle "Combined Reads at All Called PAS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFrac_myPeaksNompfilt.png
```


RNADTPlotmyPeaks_noMPFilt.sh


```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=RNADTPlotmyPeaks_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=RNADTPlotmyPeaks_noMPFilt.out
#SBATCH --error=RNADTPlotmyPeaks_noMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/rnaseq_bw/RNAseqGeuvadis_STAR_6samp_MergedBams.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand.bed -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_myPeaksNompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_myPeaksNompfilt.gz --refPointLabel "Called PAS" --plotTitle "Combined Reads at All Called PAS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_myPeaksNompfilt.png
```



I want to make one of these that look at total, nuclear, and RNA at peaks assigned to an intron. This means I need to subset the peak file to only include these. I can do this similar to how I did the UTR subset in [this analysis](pipeline_55Ind.html)  

I want to make a bedfile with these peaks. I need to also make sure they are in the final clean peaks 


makeIntronPeakBed.py
```{bash,eval=F}
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_5percCov_INTRON.bed" , "w")
okPeaks=open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_noMP_GeneLoc/Filtered_APApeaks_merged_allchrom_noMP.sort.named.noCHR_geneLocParsed.5percCov.bed", "r")


okPeak_dic={}
for ln in okPeaks:
    peak=ln.split()[3].split(":")[1]
    peak_num=peak[4:]
    okPeak_dic[peak_num]=""



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]
        if anno_lst[0].split(":")[0]=="intron":
            if peak in okPeak_dic.keys():
                peak_i=int(peak)
                start_i=int(start)
                end_i=int(end)
                type="intron"
                outFile.write("%s\t%d\t%d\t%s\t%s\t%s\n"%(chrom, start_i, end_i, type,score, strand))
    else:
        type_dic={}
        for each in anno_lst:
            type_dic[each.split(":")[0]]=each.split(":")[1]
        if "utr3" in type_dic.keys():
            continue
        if "intron" in type_dic.keys():
             if peak in okPeak_dic.keys():
                 peak_i=int(peak)
                 start_i=int(start)
                 end_i=int(end)
                 type="intron"
                 outFile.write("%s\t%d\t%d\t%s\t%s\t%s\n"%(chrom, start_i, end_i,type ,score, strand))
outFile.close()
```



BothFracDTPlotmyIntronPeaks_noMPFilt.sh
```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=BothFracDTPlotmyIntronPeaks_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=BothFracRNADTPlotmyIntronPeaks_noMPFilt.out
#SBATCH --error=BothFracRNADTPlotmyIntronPeaks_noMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/mergedBW/Total_MergedBamCoverage.bw /project2/gilad/briana/threeprimeseq/data/mergedBW/Nuclear_MergedBamCoverage.bw /project2/gilad/briana/threeprimeseq/data/rnaseq_bw/RNAseqGeuvadis_STAR_6samp_MergedBams.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand_INTRON.bed -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFrac_myPeaksIntron_Nompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFrac_myPeaksIntron_Nompfilt.gz --refPointLabel "Called Intronic PAS" --plotTitle "Combined Reads at Intronic PAS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFrac_myPeaksIntronNompfilt.png
```

RNA seq only:

RNADTPlotmyIntronPeaks_noMPFilt.sh

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=RNADTPlotmyIntronPeaks_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=RNADTPlotmyIntronPeaks_noMPFilt.out
#SBATCH --error=RNADTPlotmyIntronPeaks_noMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/rnaseq_bw/RNAseqGeuvadis_STAR_6samp_MergedBams.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand_INTRON.bed -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_myPeaksIntron_Nompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_myPeaksIntron_Nompfilt.gz --refPointLabel "Called Intronic PAS" --plotTitle "Combined Reads at Intronic PAS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_myPeaksIntronNompfilt.png
```


I should try this with the nuclear RNA samples  

I need to merge them and make a BW.  


mergeNucRNAseq.sh
```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=mergeNucRNAseq
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=mergeNucRNAseq.out
#SBATCH --error=mergeNucRNAseq.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


samtools merge /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.bam  /project2/gilad/briana/Total_Nuc_RNA_seq_data/170428_K00242_0214_AHK2GMBBXX-YG-SP20/data/sort/YG-SP20-Nuc-2_S5_L005_R1_001-sort.bam /project2/gilad/briana/Total_Nuc_RNA_seq_data/170428_K00242_0214_AHK2GMBBXX-YG-SP20/data/sort/YG-SP20-Nuc-1_S2_L005_R1_001-sort.bam

samtools sort /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.bam > /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.sort.bam

samtools index /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.sort.bam
```

NucBam2BW.sh 

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=NucBam2BW
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=NucBam2BW.out
#SBATCH --error=NucBam2BW.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

#total  
bamCoverage -b /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.sort.bam -o /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.sort.bw

```

NucRNADTPlotmyIntronPeaks_noMPFilt.sh 

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=NucRNADTPlotmyIntronPeaks_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=NucRNADTPlotmyIntronPeaks_noMPFilt.out
#SBATCH --error=NucRNADTPlotmyIntronPeaks_noMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand_INTRON.bed  -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntron_Nompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntron_Nompfilt.gz --refPointLabel "Called Intronic PAS" --plotTitle "Combined Reads at Intronic PAS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntronNompfilt.png
```

NucRNADTPlotmyIntronPeaks_noMPFilt_150.sh 

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=NucRNADTPlotmyIntronPeaks_noMPFilt_150
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=NucRNADTPlotmyIntronPeaks_noMPFilt150.out
#SBATCH --error=NucRNADTPlotmyIntronPeaks_noMPFilt150.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand_INTRON.bed  -b 150 -a 150  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntron_Nompfilt150.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntron_Nompfilt150.gz --refPointLabel "Called Intronic PAS" --plotTitle "Combined Reads at Intronic PAS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntronNompfilt150.png
```
NucRNADTPlotmyIntronPeaks_noMPFilt_300.sh 

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=NucRNADTPlotmyIntronPeaks_noMPFilt_300
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=NucRNADTPlotmyIntronPeaks_noMPFilt300.out
#SBATCH --error=NucRNADTPlotmyIntronPeaks_noMPFilt300.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/NuclearRNA/NuclearRNA_merged.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand_INTRON.bed  -b 300 -a 300  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntron_Nompfilt300.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntron_Nompfilt300.gz --refPointLabel "Called Intronic PAS" --plotTitle "Combined Reads at Intronic PAS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/NucRNA_myPeaksIntronNompfilt300.png
```


####TES and TSS (not looking right- coding end doesnt work) 

fixStrand4DTplots_TESTSS.py
Fix strand for these:  
```{bash,eval=F}
TSSIn="/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_TSSAllGenes.bed"
TESIn="/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/ncbiRefSeq_TESAllGenes.bed"
TSSOut="/project2/gilad/briana/threeprimeseq/data/peaks4DT/ncbiRefSeq_TSSAllGenes_fixedStrand.bed"
TESOut="/project2/gilad/briana/threeprimeseq/data/peaks4DT/ncbiRefSeq_TESAllGenes_fixedStrand.bed"


def fix_strand(Fin,Fout):
    fout=open(Fout,"w")
    for ln in open(Fin, "r"):
        chrom, start, end, name, score, strand = ln.split()
        if strand=="+":
            fout.write("%s\t%s\t%s\t%s\t%s\t-\n"%(chrom,start,end,name,score))
        else:
            fout.write("%s\t%s\t%s\t%s\t%s\t+\n"%(chrom,start,end,name,score))
    fout.close()
    
    
fix_strand(TSSIn, TSSOut)
fix_strand(TESIn, TESOut)
```


files to make: new TSS file from the annotation in the new gene loc annocation pipeline  

getTss.py
```{bash,eval=F}


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_TSSAllGenes.bed", "w")

for i, ln in enumerate(TXN2Gene_file):
    if i >0 :
        chrom=ln.split()[2]
        chromf=chrom[3:]
        start=int(ln.split()[6])-1 
        end=int(ln.split()[6])
        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()
```

BothFracRNADTPlotTSS_noMPFilt.sh


```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=BothFracRNADTPlotTSS_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=BothFracRNADTPlotTSS_noMPFilt.out
#SBATCH --error=BothFracRNADTPlotTSS_noMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/mergedBW/Total_MergedBamCoverage.bw /project2/gilad/briana/threeprimeseq/data/mergedBW/Nuclear_MergedBamCoverage.bw /project2/gilad/briana/threeprimeseq/data/rnaseq_bw/RNAseqGeuvadis_STAR_6samp_MergedBams.sort.bw -R /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.transcriptTSS.bed -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFracRNA_TSS_Nompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFracRNA_TSS_Nompfilt.gz --refPointLabel "Called TSS" --plotTitle "Combined Reads at TSS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFracRNA_TSS_Nompfilt.png
```


getTES.py

```{bash,eval=F}


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_TESAllGenes.bed", "w")

for i, ln in enumerate(TXN2Gene_file):
    if i >0 :
        chrom=ln.split()[2]
        chromf=chrom[3:]
        start=int(ln.split()[7])-1 
        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()
```


BothFracRNADTPlotTES_noMPFilt.sh

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=BothFracRNADTPlotTES_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=BothFracRNADTPlotTES_noMPFilt.out
#SBATCH --error=BothFracRNADTPlotTESnoMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/mergedBW/Total_MergedBamCoverage.bw /project2/gilad/briana/threeprimeseq/data/mergedBW/Nuclear_MergedBamCoverage.bw /project2/gilad/briana/threeprimeseq/data/rnaseq_bw/RNAseqGeuvadis_STAR_6samp_MergedBams.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/ncbiRefSeq_TESAllGenes_fixedStrand.bed -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFracRNA_TES_Nompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFracRNA_TES_Nompfilt.gz --refPointLabel "Called TES" --plotTitle "Combined Reads at TES" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/BothFracRNA_TES_Nompfilt.png
```


RNADTPlotTES_noMPFilt.sh

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=RNADTPlotTES_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=RNADTPlotTES_noMPFilt.out
#SBATCH --error=RNADTPlotTESnoMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/rnaseq_bw/RNAseqGeuvadis_STAR_6samp_MergedBams.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/ncbiRefSeq_TESAllGenes_fixedStrand.bed -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_TES_Nompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_TES_Nompfilt.gz --refPointLabel "Called TES" --plotTitle "Combined Reads at TES" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/RNA_TES_Nompfilt.png
```


/project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.transcriptTSS.bed

####ATAC seq 

ATAC seq bam files are in /project2/yangili1/LCL/ATAC/ I will merge the bam files.  

mergeBamFiles_ATAC.sh
```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=mergeBamFiles_ATAC
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --output=mergeBamFiles_ATAC.out
#SBATCH --error=mergeBamFiles_ATAC.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env  


samtools merge  /project2/gilad/briana/threeprimeseq/data/ATAC_mergeBam/ATAC_merged.bam /project2/yangili1/LCL/ATAC/*.sort.bam 

samtools sort /project2/gilad/briana/threeprimeseq/data/ATAC_mergeBam/ATAC_merged.bam > /project2/gilad/briana/threeprimeseq/data/ATAC_mergeBam/ATAC_merged.sort.bam

samtools index /project2/gilad/briana/threeprimeseq/data/ATAC_mergeBam/ATAC_merged.sort.bam
```


bam2BW_ATAC.sh  

```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=bam2BW_ATAC
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=bam2BW_ATAC.out
#SBATCH --error=bam2BW_ATAC.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

#total  
bamCoverage -b /project2/gilad/briana/threeprimeseq/data/ATAC_mergeBam/ATAC_merged.sort.bam -o /project2/gilad/briana/threeprimeseq/data/ATAC_mergeBW/ATAC_merged.sort.bw

```

ATACDTPlotmyIntronPeaks_noMPFilt.sh
```{bash,eval=F}
#!/bin/bash

#SBATCH --job-name=ATACDTPlotmyIntronPeaks_noMPFilt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=ATACDTPlotmyIntronPeaks_noMPFilt.out
#SBATCH --error=ATACDTPlotmyIntronPeaks_noMPFilt.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


computeMatrix reference-point -S /project2/gilad/briana/threeprimeseq/data/ATAC_mergeBW/ATAC_merged.sort.bw -R /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand_INTRON.bed  -b 1000 -a 1000  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/ATAC_myPeaksIntron_Nompfilt.gz

plotHeatmap --sortRegions descend -m /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/ATAC_myPeaksIntron_Nompfilt.gz --refPointLabel "Called Intronic PAS" --plotTitle "ATAC-seq at Intronic PAS" --heatmapHeight 7 --colorMap YlGnBu  -out /project2/gilad/briana/threeprimeseq/data/LianoglouDeepTools/ATAC_myPeaksIntronNompfilt.png
```
 
Do it with the 3' UTR peaks