Last updated: 2019-02-15
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Knit directory: threeprimeseq/analysis/
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Unstaged changes:
Modified: analysis/28ind.peak.explore.Rmd
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Modified: analysis/pipeline_55Ind.Rmd
Modified: analysis/swarmPlots_QTLs.Rmd
Modified: analysis/test.max2.Rmd
Modified: analysis/test.smash.Rmd
Modified: analysis/understandPeaks.Rmd
Modified: code/Snakefile
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These are the previous versions of the R Markdown and HTML files. If you’ve configured a remote Git repository (see ?wflow_git_remote
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File | Version | Author | Date | Message |
---|---|---|---|---|
html | bd7f203 | Briana Mittleman | 2018-07-09 | Build site. |
Rmd | 4348871 | Briana Mittleman | 2018-07-09 | call peaks on nuclear |
html | f4f1918 | Briana Mittleman | 2018-07-06 | Build site. |
Rmd | 2522654 | Briana Mittleman | 2018-07-06 | fix axis |
html | a0541e3 | Briana Mittleman | 2018-07-06 | Build site. |
Rmd | df5cfe4 | Briana Mittleman | 2018-07-06 | add Yangs peaks |
html | 9de3677 | Briana Mittleman | 2018-07-05 | Build site. |
Rmd | c619183 | Briana Mittleman | 2018-07-05 | examine long peaks |
html | 2d67ec5 | Briana Mittleman | 2018-07-05 | Build site. |
Rmd | 15c7967 | Briana Mittleman | 2018-07-05 | add split analysis |
html | 24c6663 | Briana Mittleman | 2018-07-03 | Build site. |
Rmd | 776fc62 | Briana Mittleman | 2018-07-03 | genome cov stats |
html | b48f27c | Briana Mittleman | 2018-07-02 | Build site. |
Rmd | 1e2ff4c | Briana Mittleman | 2018-07-02 | evaluate bedgraph regions |
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.
#!/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.
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.
#!/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.
#!/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.
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
#!/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.
library(dplyr)
Attaching package: 'dplyr'
The following objects are masked from 'package:stats':
filter, lag
The following objects are masked from 'package:base':
intersect, setdiff, setequal, union
library(ggplot2)
library(readr)
library(workflowr)
This is workflowr version 1.2.0
Run ?workflowr for help getting started
library(tidyr)
First I will look at the bedgraph file. This is not as imformative becuase it combined regions with the same counts.
total_bedgraph=read.table("../data/bedgraph_peaks/TotalBamFiles_no0.peaks.bed",col.names = c("chr", "start", "end", "regions", "mean", "counts"))
Plot the mean:
plot(sort(log10(total_bedgraph$mean), decreasing=T), xlab="Region", ylab="log10 of bedgraph region bin", main="Distribution of log10 region means from bedgraph")
Version | Author | Date |
---|---|---|
24c6663 | Briana Mittleman | 2018-07-03 |
I want to look at the distribution of how many bases are included in the regions.
Tregion_bases=total_bedgraph %>% mutate(bases=end-start) %>% select(bases)
plot(sort(log10(Tregion_bases$bases), decreasing = T), xlab="Region", ylab="log10 of region size", main="Distribution of bases in regions- log10")
Version | Author | Date |
---|---|---|
24c6663 | Briana Mittleman | 2018-07-03 |
Given the reads are abotu 60bp this is probably pretty good.
I am only going to look at the number of bases in region and mean coverage columns here because the file is really big.
total_gencov=read.table("../data/bedgraph_peaks/TotalBamFiles.gencovpeaks_noregstring.bed",col.names = c("chr", "start", "end", "regions", "mean"))
Plot the mean:
plot(sort(log10(total_gencov$mean), decreasing=T), xlab="Region", ylab="log10 of mean bin count", main="Distribution of log10 region means")
Version | Author | Date |
---|---|---|
24c6663 | Briana Mittleman | 2018-07-03 |
plot(sort(log10(total_gencov$regions), decreasing = T), xlab="Region", ylab="log10 of region size", main="Distribution of bases in regions- log10")
Version | Author | Date |
---|---|---|
24c6663 | Briana Mittleman | 2018-07-03 |
Plot number of bases against the mean:
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")
Version | Author | Date |
---|---|---|
24c6663 | Briana Mittleman | 2018-07-03 |
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
#!/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.
awk '{if ($3 != 0) print}' TotalBamFiles.split.genomecov.bed > TotalBamFiles.split.genomecov.no0.bed
awk '{print $1 "\t" $2 "\t" $2 "\t" $3}' TotalBamFiles.split.genomecov.no0.bed > TotalBamFiles.split.genomecov.no0.fixed.bed
Use this file to run mergeGencov.sh.
total_gencov_split=read.table("../data/bedgraph_peaks/TotalBamFiles.split.gencovpeaks.noregstring.bed",col.names = c("chr", "start", "end", "regions", "mean"))
Plot the region size. I expect some of the long regions are gone.
plot(sort(log10(total_gencov_split$regions), decreasing = T), xlab="Region", ylab="log10 of region size", main="Distribution of bases in regions- log10 SPLIT")
Version | Author | Date |
---|---|---|
2d67ec5 | Briana Mittleman | 2018-07-05 |
Plot the region size against the mean:
Plot number of bases against the mean:
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")
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:
grep -n 65266512 TotalBamFiles.split.gencovpeaks.bed | awk '{print $6}' > loc_ch11_65266512_65268654.txt
loc_ch11_65266512_65268654=read.csv("../data/bedgraph_peaks/loc_ch11_65266512_65268654.txt", header=F) %>% t
loc_ch11_65266512_65268654_df= as.data.frame(loc_ch11_65266512_65268654)
loc_ch11_65266512_65268654_df$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")
Version | Author | Date |
---|---|---|
9de3677 | Briana Mittleman | 2018-07-05 |
Try one more. Example. line 816811, chr:17- 79476983- 79477761
grep -n 79476983 TotalBamFiles.split.gencovpeaks.bed | awk '{print $6}' > loc_ch17_79476983_79477761.txt
loc_ch17_79476983_79477761=read.csv("../data/bedgraph_peaks/loc_ch17_79476983_79477761.txt", header=F) %>% t
loc_ch17_79476983_79477761_df= as.data.frame(loc_ch17_79476983_79477761)
loc_ch17_79476983_79477761_df$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")
Version | Author | Date |
---|---|---|
9de3677 | Briana Mittleman | 2018-07-05 |
This one is not multiple peaks but it does need to be trimmed.
Yang created an adhoc method to do this.
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.
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
plot(sort(log10(YL_peaks$length), decreasing = T), xlab="Region", ylab="log10 of region size", main="Distribution of bases in YL regions- log10 ")
Version | Author | Date |
---|---|---|
a0541e3 | Briana Mittleman | 2018-07-06 |
Plot number of bases against the mean:
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")
library(cowplot)
Attaching package: 'cowplot'
The following object is masked from 'package:ggplot2':
ggsave
plot_grid(splitplot, YLplot)
#!/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.
#!/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:
cat * | sort -k 1,1 -k2,2n > APApeaks_nuclear_all.bed
Thoughts:
Remove peaks outside 1kb of the genes
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] cowplot_0.9.3 bindrcpp_0.2.2 tidyr_0.8.1 workflowr_1.2.0
[5] readr_1.1.1 ggplot2_3.0.0 dplyr_0.7.6
loaded via a namespace (and not attached):
[1] Rcpp_0.12.19 pillar_1.3.0 compiler_3.5.1 git2r_0.24.0
[5] plyr_1.8.4 bindr_0.1.1 tools_3.5.1 digest_0.6.17
[9] jsonlite_1.6 lattice_0.20-35 evaluate_0.13 tibble_1.4.2
[13] gtable_0.2.0 pkgconfig_2.0.2 rlang_0.2.2 Matrix_1.2-14
[17] yaml_2.2.0 withr_2.1.2 stringr_1.4.0 knitr_1.20
[21] fs_1.2.6 hms_0.4.2 rprojroot_1.3-2 grid_3.5.1
[25] tidyselect_0.2.4 reticulate_1.10 glue_1.3.0 R6_2.3.0
[29] rmarkdown_1.11 purrr_0.2.5 magrittr_1.5 whisker_0.3-2
[33] backports_1.1.2 scales_1.0.0 htmltools_0.3.6 MASS_7.3-50
[37] assertthat_0.2.0 colorspace_1.3-2 labeling_0.3 stringi_1.2.4
[41] lazyeval_0.2.1 munsell_0.5.0 crayon_1.3.4