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deNovopeakcalling.Rmd
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deNovopeakcalling.Rmd
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---
title: "deNovo peak callling"
author: "Briana Mittleman"
date: "6/28/2018"
output: html_document
---
```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = TRUE)
```
##Create Bedgraph
I will call peaks de novo in the combined total and nuclear fraction 3' Seq. The data is reletevely clean so I will start with regions that have continuous coverage. I will first create a bedgraph.
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=Tbedgraph
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=Tbedgraph.out
#SBATCH --error=Tbedgraph.err
#SBATCH --partition=broadwl
#SBATCH --mem=40G
#SBATCH --mail-type=END
module load Anaconda3
source activate three-prime-env
samtools sort -o /project2/gilad/briana/threeprimeseq/data/macs2/TotalBamFiles.sort.bam /project2/gilad/briana/threeprimeseq/data/macs2/TotalBamFiles.bam
bedtools genomecov -ibam /project2/gilad/briana/threeprimeseq/data/macs2/TotalBamFiles.sort.bam -bga > /project2/gilad/briana/threeprimeseq/data/bedgraph/TotalBamFiles.bedgraph
```
Next I will create the file without the 0 places in the genome. I will be able to use this for the bedtools merge function.
```{bash, eval=F}
awk '{if ($4 != 0) print}' TotalBamFiles.bedgraph >TotalBamFiles_no0.bedgraph
```
I can merge the regions with consequtive reads using the bedtools merge function.
* -i input bed
* -c colomn to act on
* -o collapse, print deliminated list of the counts from -c call
* -delim ","
This is the mergeBedgraph.sh script. It takes in the no 0 begraph filename without the path.
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=merge
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --output=merge.out
#SBATCH --error=merge.err
#SBATCH --partition=broadwl
#SBATCH --mem=16G
#SBATCH --mail-type=END
module load Anaconda3
source activate three-prime-env
bedgraph=$1
describer=$(echo ${bedgraph} | sed -e "s/.bedgraph$//")
bedtools merge -c 4,4,4 -o count,mean,collapse -delim "," -i /project2/gilad/briana/threeprimeseq/data/bedgraph/$1 > /project2/gilad/briana/threeprimeseq/data/bedgraph/${describer}.peaks.bed
```
Run this first on the total bedgraph, TotalBamFiles_no0.bedgraph. The file has chromosome, start, end, number of regions, mean, and a string of the values.
This is not exaclty what I want. I need to go back and do genome cov not collapsing with bedgraph.
To evaluate this I will bring the file into R and plot some statistics about it.
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=Tgencov
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=Tgencov.out
#SBATCH --error=Tgencov.err
#SBATCH --partition=broadwl
#SBATCH --mem=40G
#SBATCH --mail-type=END
module load Anaconda3
source activate three-prime-env
bedtools genomecov -ibam /project2/gilad/briana/threeprimeseq/data/macs2/TotalBamFiles.sort.bam -d > /project2/gilad/briana/threeprimeseq/data/bedgraph/TotalBamFiles.genomecov.bed
```
I will now remove the bases with 0 coverage.
```{bash, eval=F}
awk '{if ($3 != 0) print}' TotalBamFiles.genomecov.bed > TotalBamFiles.genomecov.no0.bed
awk '{print $1 "\t" $2 "\t" $2 "\t" $3}' TotalBamFiles.genomecov.no0.bed > TotalBamFiles.genomecov.no0.fixed.bed
```
I will now merge the genomecov_no0 file with mergeGencov.sh
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=mergegc
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --output=mergegc.out
#SBATCH --error=mergegc.err
#SBATCH --partition=broadwl
#SBATCH --mem=16G
#SBATCH --mail-type=END
module load Anaconda3
source activate three-prime-env
gencov=$1
describer=$(echo ${gencov} | sed -e "s/.genomecov.no0.fixed.bed$//")
bedtools merge -c 4,4,4 -o count,mean,collapse -delim "," -i /project2/gilad/briana/threeprimeseq/data/bedgraph/$1 > /project2/gilad/briana/threeprimeseq/data/bedgraph/${describer}.gencovpeaks.bed
```
This method gives us 811,637 regions.
##Evaluate regions
###Bedgraph results
```{r}
library(dplyr)
library(ggplot2)
library(readr)
library(workflowr)
library(tidyr)
```
First I will look at the bedgraph file. This is not as imformative becuase it combined regions with the same counts.
```{r}
total_bedgraph=read.table("../data/bedgraph_peaks/TotalBamFiles_no0.peaks.bed",col.names = c("chr", "start", "end", "regions", "mean", "counts"))
```
Plot the mean:
```{r}
plot(sort(log10(total_bedgraph$mean), decreasing=T), xlab="Region", ylab="log10 of bedgraph region bin", main="Distribution of log10 region means from bedgraph")
```
I want to look at the distribution of how many bases are included in the regions.
```{r}
Tregion_bases=total_bedgraph %>% mutate(bases=end-start) %>% select(bases)
plot(sort(log10(Tregion_bases$bases), decreasing = T), xlab="Region", ylab="log10 of region size", main="Distribution of bases in regions- log10")
```
Given the reads are abotu 60bp this is probably pretty good.
###GenomeCov results
I am only going to look at the number of bases in region and mean coverage columns here because the file is really big.
```{r}
total_gencov=read.table("../data/bedgraph_peaks/TotalBamFiles.gencovpeaks_noregstring.bed",col.names = c("chr", "start", "end", "regions", "mean"))
```
Plot the mean:
```{r}
plot(sort(log10(total_gencov$mean), decreasing=T), xlab="Region", ylab="log10 of mean bin count", main="Distribution of log10 region means")
```
```{r}
plot(sort(log10(total_gencov$regions), decreasing = T), xlab="Region", ylab="log10 of region size", main="Distribution of bases in regions- log10")
```
Plot number of bases against the mean:
```{r}
ggplot(total_gencov, aes(y=log10(regions), x=log10(mean))) +
geom_point(na.rm = TRUE, size = 0.1) +
geom_density2d(na.rm = TRUE, size = 1, colour = 'red') +
ylab('Log10 Region size') +
xlab('Log10 Mean region coverage') +
ggtitle("Region size vs Region Coverage: Combined Total Libraries")
```
##Troubleshooting
###Account for split reads
In the previous analysis I did not account for split reads in the genome coveragre step. This may explain some of the long regions that are an effect of splicing. This script is
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=Tgencovsplit
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=Tgencovsplit.out
#SBATCH --error=Tgencovaplit.err
#SBATCH --partition=broadwl
#SBATCH --mem=40G
#SBATCH --mail-type=END
module load Anaconda3
source activate three-prime-env
bedtools genomecov -ibam /project2/gilad/briana/threeprimeseq/data/macs2/TotalBamFiles.sort.bam -d -split > /project2/gilad/briana/threeprimeseq/data/bedgraph/TotalBamFiles.split.genomecov.bed
```
Now I need to remove the 0s and merge.
```{bash, eval=F}
awk '{if ($3 != 0) print}' TotalBamFiles.split.genomecov.bed > TotalBamFiles.split.genomecov.no0.bed
awk '{print $1 "\t" $2 "\t" $2 "\t" $3}' TotalBamFiles.split.genomecov.no0.bed > TotalBamFiles.split.genomecov.no0.fixed.bed
```
Use this file to run mergeGencov.sh.
```{r}
total_gencov_split=read.table("../data/bedgraph_peaks/TotalBamFiles.split.gencovpeaks.noregstring.bed",col.names = c("chr", "start", "end", "regions", "mean"))
```
Plot the region size. I expect some of the long regions are gone.
```{r}
plot(sort(log10(total_gencov_split$regions), decreasing = T), xlab="Region", ylab="log10 of region size", main="Distribution of bases in regions- log10 SPLIT")
```
Plot the region size against the mean:
Plot number of bases against the mean:
```{r}
splitplot=ggplot(total_gencov_split, aes(y=log10(regions), x=log10(mean))) +
geom_point(na.rm = TRUE, size = 0.1) +
geom_density2d(na.rm = TRUE, size = 1, colour = 'red') +
ylab('Log10 Region size') +
xlab('Log10 Mean region coverage') +
scale_y_continuous(limits = c(0, 3)) +
ggtitle("Combined Total Libraries SPLIT")
```
###Investigate long regions
Some of the regions are long and probably represent 2 or more sites. This is evident in highly expressed genes such as actB. I will look at some of the long regions and make histograms with the strings of coverage in the region.
First I am going to look at chr11:65266512-65268654, this is peak 580475 I will go into the otalBamFiles.split.gencovpeaks.bed file and use:
```{bash, eval=F}
grep -n 65266512 TotalBamFiles.split.gencovpeaks.bed | awk '{print $6}' > loc_ch11_65266512_65268654.txt
```
```{r}
loc_ch11_65266512_65268654=read.csv("../data/bedgraph_peaks/loc_ch11_65266512_65268654.txt", header=F) %>% t
loc_ch11_65266512_65268654_df= as.data.frame(loc_ch11_65266512_65268654)
loc_ch11_65266512_65268654_df$loc= seq(1:nrow(loc_ch11_65266512_65268654_df))
colnames(loc_ch11_65266512_65268654_df)= c("count", "loc")
ggplot(loc_ch11_65266512_65268654_df, aes(x=loc, y=count)) + geom_line() + labs(y="Read Count", x="Peak Location", title="Example of long region called as 1 peak \n ch11 65266512-65268654")
```
Try one more. Example. line 816811, chr:17- 79476983- 79477761
```{bash, eval=F}
grep -n 79476983 TotalBamFiles.split.gencovpeaks.bed | awk '{print $6}' > loc_ch17_79476983_79477761.txt
```
```{r}
loc_ch17_79476983_79477761=read.csv("../data/bedgraph_peaks/loc_ch17_79476983_79477761.txt", header=F) %>% t
loc_ch17_79476983_79477761_df= as.data.frame(loc_ch17_79476983_79477761)
loc_ch17_79476983_79477761_df$loc= seq(1:nrow(loc_ch17_79476983_79477761_df))
colnames(loc_ch17_79476983_79477761_df)= c("count", "loc")
ggplot(loc_ch17_79476983_79477761_df, aes(x=loc, y=count)) + geom_line() + labs(y="Read Count", x="Peak Location", title="Example of long region called as 1 peak \n ch17 79476983:79477761")
```
This one is not multiple peaks but it does need to be trimmed.
##Compare to adhoc method by Yang
Yang created an adhoc method to do this.
```{python, eval=F}
def main(inFile, outFile, ctarget):
fout = open(outFile,'w')
mincount = 10
ov = 20
current_peak = []
currentChrom = None
for ln in open(inFile):
chrom, pos, count = ln.split()
if chrom != ctarget: continue
count = float(count)
if currentChrom == None:
currentChrom = chrom
if count == 0 or currentChrom != chrom:
if len(current_peak) > 0:
M = max([x[1] for x in current_peak])
if M > mincount:
all_peaks = refine_peak(current_peak, M, M*0.1,M*0.05)
#refined_peaks = [(x[0][0],x[-1][0], np.mean([y[1] for y in x])) for x in all_peaks]
rpeaks = [(int(x[0][0])-ov,int(x[-1][0])+ov, np.mean([y[1] for y in x])) for x in all_peaks]
if len(rpeaks) > 1:
for clu in cluster_intervals(rpeaks)[0]:
M = max([x[2] for x in clu])
merging = []
for x in clu:
if x[2] > M *0.5:
#print x, M
merging.append(x)
c, s,e,mean = chrom, min([x[0] for x in merging])+ov, max([x[1] for x in merging])-ov, np.mean([x[2] for x in merging])
#print c,s,e,mean
fout.write("chr%s\t%d\t%d\t%d\t+\t.\n"%(c,s,e,mean))
fout.flush()
elif len(rpeaks) == 1:
s,e,mean = rpeaks[0]
fout.write("chr%s\t%d\t%d\t%f\t+\t.\n"%(chrom,s+ov,e-ov,mean))
print ("chr%s"%chrom+"\t%d\t%d\t%f\t+\t.\n"%rpeaks[0])
#print refined_peaks
current_peak = []
else:
current_peak.append((pos,count))
currentChrom = chrom
def refine_peak(current_peak, M, thresh, noise, minpeaksize=30):
cpeak = []
opeak = []
allcpeaks = []
allopeaks = []
for pos, count in current_peak:
if count > thresh:
cpeak.append((pos,count))
opeak = []
continue
elif count > noise:
opeak.append((pos,count))
else:
if len(opeak) > minpeaksize:
allopeaks.append(opeak)
opeak = []
if len(cpeak) > minpeaksize:
allcpeaks.append(cpeak)
cpeak = []
if len(cpeak) > minpeaksize:
allcpeaks.append(cpeak)
if len(opeak) > minpeaksize:
allopeaks.append(opeak)
allpeaks = allcpeaks
for opeak in allopeaks:
M = max([x[1] for x in opeak])
allpeaks += refine_peak(opeak, M, M*0.3, noise)
#print [(x[0],x[-1]) for x in allcpeaks], [(x[0],x[-1]) for x in allopeaks], [(x[0],x[-1]) for x in allpeaks]
#print '---\n'
return allpeaks
if __name__ == "__main__":
import numpy as np
from misc_helper import *
import sys
chrom = sys.argv[1]
inFile = "/project2/yangili1/threeprimeseq/gencov/TotalBamFiles.split.genomecov.bed"
outFile = "APApeaks_chr%s.bed"%chrom
main(inFile, outFile, chrom)
```
This is done by chromosome and takes in the TotalBam Split genome coverage file I made.I am going to look at the stats for these peaks.
```{r}
YL_peaks=read.table("../data/bedgraph_peaks/APApeaks.bed", col.names = c("chr", "start", "end", "count", "strand", "score")) %>% mutate(length=end-start)
```
Plot the lengths
```{r}
plot(sort(log10(YL_peaks$length), decreasing = T), xlab="Region", ylab="log10 of region size", main="Distribution of bases in YL regions- log10 ")
```
Plot number of bases against the mean:
```{r}
YLplot=ggplot(YL_peaks, aes(y=log10(length), x=log10(count))) +
geom_point(na.rm = TRUE, size = 0.1) +
geom_density2d(na.rm = TRUE, size = 1, colour = 'red') +
ylab('Log10 Region size') +
xlab('Log10 Mean region coverage') +
scale_y_continuous(limits = c(0, 3)) +
ggtitle("YL Peaks Combined Total Libraries")
```
```{r}
library(cowplot)
plot_grid(splitplot, YLplot)
```
##Run this on the Nuclear Fraction Bam
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=Ngencov_s
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=Ngencov_s.out
#SBATCH --error=Ngencov_s.err
#SBATCH --partition=broadwl
#SBATCH --mem=40G
#SBATCH --mail-type=END
module load Anaconda3
source activate three-prime-env
samtools sort -o /project2/gilad/briana/threeprimeseq/data/macs2/NuclearBamFiles.sort.bam /project2/gilad/briana/threeprimeseq/data/macs2/NuclearBamFiles.bam
bedtools genomecov -ibam /project2/gilad/briana/threeprimeseq/data/macs2/NuclearBamFiles.sort.bam -d -split > /project2/gilad/briana/threeprimeseq/data/bedgraph/NuclearBamFiles.split.genomecov.bed
```
I modified Yang's script to take the nuclear gencov and put the output in the data/peaks directory. I will create a wrapper to call this on chromosomes 1-22.
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=w_getpeakYL
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=w_getpeakYL.out
#SBATCH --error=w_getpeakYL.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END
module load Anaconda3
source activate three-prime-env
for i in $(seq 1 22); do
sbatch callPeaksYL_Nuc.py $i
done
```
I can now concatenate all of these into one file:
```{bash, eval=F}
cat * | sort -k 1,1 -k2,2n > APApeaks_nuclear_all.bed
```
Thoughts:
* Remove peaks outside 1kb of the genes
* Remove peaks with low expression