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smash_smooth.Rmd
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smash_smooth.Rmd
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---
title: "SMASH_smooth"
author: "Briana Mittleman"
date: "7/19/2018"
output: html_document
---
```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = TRUE)
```
Use SMASH to smooth regions of the Netseq data.
I want to use the first 8 lines (NET3)
* 18505
* 18508
* 18486
* 19239
* 19141
* 19193
* 19257
* 19128
The coverage files I need to make the matrix are in /project2/gilad/briana/Net-seq-pilot/data/cov.
##ACTB
I am going to look at the region chr7:5,565,291-5,573,483 around the actb gene.
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=testsmash.actb
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=testsmash.actb.out
#SBATCH --error=testsmash.actb.err
#SBATCH --partition=broadwl
#SBATCH --mem=20G
#SBATCH --mail-type=END
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-18486_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-18486_combined_Netpilot-actb.bed
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-18505_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-18505_combined_Netpilot-actb.bed
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-18508_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-18508_combined_Netpilot-actb.bed
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19128_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19128_combined_Netpilot-sort.actb.bed
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19141_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19141_combined_Netpilot.actb.bed
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19193_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19193_combined_Netpilot.actb.bed
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19239_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19239_combined_Netpilot.actb.bed
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19257_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19257_combined_Netpilot.actb.bed
```
Load Packages:
```{r}
library(devtools)
library(scales)
library(smashr)
library(tidyr)
library(workflowr)
library(dplyr)
```
```{r}
actb_18486=read.table("../data/smash_test/YG-SP-NET3-18486_combined_Netpilot-actb.bed", col.names=c("chr", "base", "net18486"))
actb_18505=read.table("../data/smash_test/YG-SP-NET3-18505_combined_Netpilot-actb.bed", col.names=c("chr", "base", "net18505"))
actb_18508=read.table("../data/smash_test/YG-SP-NET3-18508_combined_Netpilot-actb.bed", col.names=c("chr", "base", "net18508"))
actb_19128=read.table("../data/smash_test/YG-SP-NET3-19128_combined_Netpilot.actb.bed", col.names=c("chr", "base", "net19128"))
actb_19141=read.table("../data/smash_test/YG-SP-NET3-19141_combined_Netpilot.actb.bed", col.names=c("chr", "base", "net19141"))
actb_19193=read.table("../data/smash_test/YG-SP-NET3-19193_combined_Netpilot.actb.bed", col.names=c("chr", "base", "net19193"))
actb_19239=read.table("../data/smash_test/YG-SP-NET3-19239_combined_Netpilot.actb.bed", col.names=c("chr", "base", "net19239"))
actb_19257=read.table("../data/smash_test/YG-SP-NET3-19257_combined_Netpilot.actb.bed", col.names=c("chr", "base", "net19257"))
```
Make matrix
```{r}
actb_matrix=cbind(actb_18486$net18486, actb_18505$net18505,actb_18508$net18508, actb_19128$net19128, actb_19141$net19141,actb_19193$net19193, actb_19239$net19239, actb_19257$net19257) %>%t
```
write the matrix out:
```{r}
#write.table(actb_matrix, "../data/smash_test/actb_countmatrix.txt", quote = F, row.names = F, col.names = F)
```
Run smash:
```{r}
actb_res = smash.poiss(actb_matrix[1,]+actb_matrix[2,]+ actb_matrix[3,]+ actb_matrix[4,]+actb_matrix[5,]+actb_matrix[6,]+actb_matrix[7,]+actb_matrix[8,], post.var=TRUE)
```
```{r}
region=5565291:5573483
plot(region,actb_res$est,type='l',xlab="position",ylab="intensity", main="SMASH results ACTB")
```
Next I want to run it seperatly for all 8 individuals and plot them together to see if the peaks line up.
```{r}
actb_res1 = smash.poiss(actb_matrix[1,], post.var=TRUE)
actb_res2 = smash.poiss(actb_matrix[2,], post.var=TRUE)
actb_res3 = smash.poiss(actb_matrix[3,], post.var=TRUE)
actb_res4 = smash.poiss(actb_matrix[4,], post.var=TRUE)
actb_res5 = smash.poiss(actb_matrix[5,], post.var=TRUE)
actb_res6 = smash.poiss(actb_matrix[6,], post.var=TRUE)
actb_res7 = smash.poiss(actb_matrix[7,], post.var=TRUE)
actb_res8 = smash.poiss(actb_matrix[8,], post.var=TRUE)
```
Make results matrix:
```{r}
res_matrix=rbind(actb_res$est,actb_res1$est,actb_res2$est,actb_res3$est,actb_res4$est,actb_res5$est,actb_res6$est,actb_res7$est,actb_res8$est)
#write.table(res_matrix, "../data/smash_test/actb_smash_res.txt", quote = F, col.names = F, row.names = F, sep="\t")
```
```{r}
par(mfrow=c(3,3))
plot(region,actb_res$est,type='l',xlab="position",ylab="intensity", main="Combined")
plot(region,actb_res1$est,type='l',xlab="position",ylab="intensity", main="18486")
plot(region,actb_res2$est,type='l',xlab="position",ylab="intensity",main="18505")
plot(region,actb_res3$est,type='l',xlab="position",ylab="intensity",main="18508")
plot(region,actb_res4$est,type='l',xlab="position",ylab="intensity",main="19128")
plot(region,actb_res5$est,type='l',xlab="position",ylab="intensity",main="19141")
plot(region,actb_res6$est,type='l',xlab="position",ylab="intensity",main="19193")
plot(region,actb_res7$est,type='l',xlab="position",ylab="intensity",main="19239")
plot(region,actb_res8$est,type='l',xlab="position",ylab="intensity",main="19257")
```
I also am going to run it on the Mayer hek data.
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=testsmash.actb
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=testsmash.actb.out
#SBATCH --error=testsmash.actb.err
#SBATCH --partition=broadwl
#SBATCH --mem=20G
#SBATCH --mail-type=END
awk '$1 == 7 && $2 >= 5565291 && $2 <= 5573483 {print}' /project2/gilad/briana/mayer.data/mayer_hek/data/cov/mayer_hek-sort.dedup.cov.bed > /project2/gilad/briana/mayer.data/mayer_hek/data/mayer_hek-dedup-smashactb.bed
```
This is not the correct coverage file to run this on. I need to make a genome coverage file of the data then I can run this.
##COMT
Second gene to look at COMT chr22:19,928,209-19,960,444
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=testsmash.comt
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=testsmash.comt.out
#SBATCH --error=testsmash.comt.err
#SBATCH --partition=broadwl
#SBATCH --mem=20G
#SBATCH --mail-type=END
awk '$1 == 22 && $2 >= 19928209 && $2 <= 19960444 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-18486_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-18486_combined_Netpilot.comt.bed
awk '$1 == 22 && $2 >= 19928209 && $2 <= 19960444 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-18505_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-18505_combined_Netpilot.comt.bed
awk '$1 == 22 && $2 >= 19928209 && $2 <= 19960444 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-18508_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-18508_combined_Netpilot.comt.bed
awk '$1 == 22 && $2 >= 19928209 && $2 <= 19960444 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19128_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19128_combined_Netpilot.comt.bed
awk '$1 == 22 && $2 >= 19928209 && $2 <= 19960444 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19141_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19141_combined_Netpilot.comt.bed
awk '$1 == 22 && $2 >= 19928209 && $2 <= 19960444 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19193_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19193_combined_Netpilot.comt.bed
awk '$1 == 22 && $2 >= 19928209 && $2 <= 19960444 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19239_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19239_combined_Netpilot.comt.bed
awk '$1 == 22 && $2 >= 19928209 && $2 <= 19960444 {print}' /project2/gilad/briana/Net-seq-pilot/data/cov/YG-SP-NET3-19257_combined_Netpilot-sort.cov.bed > /project2/gilad/briana/Net-seq-pilot/data/smash.test/YG-SP-NET3-19257_combined_Netpilot.comt.bed
```
```{r}
comt_18486=read.table("../data/smash_test/YG-SP-NET3-18486_combined_Netpilot-comt.bed", col.names=c("chr", "base", "net18486"))
comt_18505=read.table("../data/smash_test/YG-SP-NET3-18505_combined_Netpilot-comt.bed", col.names=c("chr", "base", "net18505"))
comt_18508=read.table("../data/smash_test/YG-SP-NET3-18508_combined_Netpilot-comt.bed", col.names=c("chr", "base", "net18508"))
comt_19128=read.table("../data/smash_test/YG-SP-NET3-19128_combined_Netpilot-comt.bed", col.names=c("chr", "base", "net19128"))
comt_19141=read.table("../data/smash_test/YG-SP-NET3-19141_combined_Netpilot.comt.bed", col.names=c("chr", "base", "net19141"))
comt_19193=read.table("../data/smash_test/YG-SP-NET3-19193_combined_Netpilot-comt.bed", col.names=c("chr", "base", "net19193"))
comt_19239=read.table("../data/smash_test/YG-SP-NET3-19239_combined_Netpilot.comt.bed", col.names=c("chr", "base", "net19239"))
comt_19257=read.table("../data/smash_test/YG-SP-NET3-19257_combined_Netpilot.comt.bed", col.names=c("chr", "base", "net19257"))
```
Make matrix
```{r}
comt_matrix=cbind(comt_18486$net18486, comt_18505$net18505,comt_18508$net18508, comt_19128$net19128, comt_19141$net19141,comt_19193$net19193, comt_19239$net19239, comt_19257$net19257) %>%t
```
write the matrix out:
```{r}
#write.table(comt_matrix, "../data/smash_test/comt_countmatrix.txt", quote = F, row.names = F, col.names = F)
```
Run smash:
```{r}
comt_res = smash.poiss(comt_matrix[1,]+comt_matrix[2,]+ comt_matrix[3,]+ comt_matrix[4,]+comt_matrix[5,]+comt_matrix[6,]+comt_matrix[7,]+comt_matrix[8,], post.var=TRUE)
comt_res1 = smash.poiss(comt_matrix[1,], post.var=TRUE)
comt_res2 = smash.poiss(comt_matrix[2,], post.var=TRUE)
comt_res3 = smash.poiss(comt_matrix[3,], post.var=TRUE)
comt_res4 = smash.poiss(comt_matrix[4,], post.var=TRUE)
comt_res5 = smash.poiss(comt_matrix[5,], post.var=TRUE)
comt_res6 = smash.poiss(comt_matrix[6,], post.var=TRUE)
comt_res7 = smash.poiss(comt_matrix[7,], post.var=TRUE)
comt_res8 = smash.poiss(comt_matrix[8,], post.var=TRUE)
```
Make results matrix:
```{r}
res_matrix_comt=rbind(comt_res$est,comt_res1$est,comt_res2$est,comt_res3$est,comt_res4$est,comt_res5$est,comt_res6$est,comt_res7$est,comt_res8$est)
#write.table(res_matrix_comt, "../data/smash_test/comt_smash_res.txt", quote = F, col.names = F, row.names = F, sep="\t")
```
```{r}
comtregion=19928209:19960444
par(mfrow=c(3,3))
plot(comtregion,comt_res$est,type='l',xlab="position",ylab="intensity", main="Combined")
plot(comtregion,comt_res1$est,type='l',xlab="position",ylab="intensity", main="18486")
plot(comtregion,comt_res2$est,type='l',xlab="position",ylab="intensity",main="18505")
plot(comtregion,comt_res3$est,type='l',xlab="position",ylab="intensity",main="18508")
plot(comtregion,comt_res4$est,type='l',xlab="position",ylab="intensity",main="19128")
plot(comtregion,comt_res5$est,type='l',xlab="position",ylab="intensity",main="19141")
plot(comtregion,comt_res6$est,type='l',xlab="position",ylab="intensity",main="19193")
plot(comtregion,comt_res7$est,type='l',xlab="position",ylab="intensity",main="19239")
plot(comtregion,comt_res8$est,type='l',xlab="position",ylab="intensity",main="19257")
```
I will next subset the bam files for each of these regions to create bigwig files that can be uploaded to UCSC.
```{bash, eval=F}
#!/bin/bash
#SBATCH --job-name=subset.actb
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=subset.actb.out
#SBATCH --error=subset.actb.err
#SBATCH --partition=broadwl
#SBATCH --mem=20G
#SBATCH --mail-type=END
module load Anaconda3
source activate net-seq
samtools view -bh /project2/gilad/briana/Net-seq-pilot/data/sort/YG-SP-NET3-18486_combined_Netpilot-sort.bam 7:5565291-5573483 > /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-18486_combined_Netpilot.actb.bam
samtools view -bh /project2/gilad/briana/Net-seq-pilot/data/sort/YG-SP-NET3-18505_combined_Netpilot-sort.bam 7:5565291-5573483 > /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-18505_combined_Netpilot.actb.bam
samtools view -bh /project2/gilad/briana/Net-seq-pilot/data/sort/YG-SP-NET3-18508_combined_Netpilot-sort.bam 7:5565291-5573483 > /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-18508_combined_Netpilot.actb.bam
samtools view -bh /project2/gilad/briana/Net-seq-pilot/data/sort/YG-SP-NET3-19128_combined_Netpilot-sort.bam 7:5565291-5573483 > /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19128_combined_Netpilot.actb.bam
samtools view -bh /project2/gilad/briana/Net-seq-pilot/data/sort/YG-SP-NET3-19141_combined_Netpilot-sort.bam 7:5565291-5573483 > /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19141_combined_Netpilot.actb.bam
samtools view -bh /project2/gilad/briana/Net-seq-pilot/data/sort/YG-SP-NET3-19193_combined_Netpilot-sort.bam 7:5565291-5573483 > /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19193_combined_Netpilot.actb.bam
samtools view -bh /project2/gilad/briana/Net-seq-pilot/data/sort/YG-SP-NET3-19239_combined_Netpilot-sort.bam 7:5565291-5573483 > /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19239_combined_Netpilot.actb.bam
samtools view -bh /project2/gilad/briana/Net-seq-pilot/data/sort/YG-SP-NET3-19257_combined_Netpilot-sort.bam 7:5565291-5573483 > /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19257_combined_Netpilot.actb.bam
```
Make Bigwig using deepools command:
```{bash, eval=FALSE}
#!/bin/bash
#SBATCH --job-name=actb_bigwig
#SBATCH --time=8:00:00
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=actb_bigwig.out
#SBATCH --error=act_bigmost.err
#SBATCH --partition=broadwl
#SBATCH --mem=20G
#SBATCH --mail-type=END
module load Anaconda3
source activate net-seq
bamCoverage -b /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-18486_combined_Netpilot.actb.bam -o /project2/gilad/briana/Net-seq-pilot/data/bigwig/YG-SP-NET3-18486_combined_Netpilot.actb.bw
bamCoverage -b /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-18505_combined_Netpilot.actb.bam -o /project2/gilad/briana/Net-seq-pilot/data/bigwig/YG-SP-NET3-18505_combined_Netpilot.actb.bw
bamCoverage -b /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-18508_combined_Netpilot.actb.bam -o /project2/gilad/briana/Net-seq-pilot/data/bigwig/YG-SP-NET3-18508_combined_Netpilot.actb.bw
bamCoverage -b /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19128_combined_Netpilot.actb.bam -o /project2/gilad/briana/Net-seq-pilot/data/bigwig/YG-SP-NET3-19128_combined_Netpilot.actb.bw
bamCoverage -b /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19141_combined_Netpilot.actb.bam -o /project2/gilad/briana/Net-seq-pilot/data/bigwig/YG-SP-NET3-19141_combined_Netpilot.actb.bw
bamCoverage -b /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19193_combined_Netpilot.actb.bam -o /project2/gilad/briana/Net-seq-pilot/data/bigwig/YG-SP-NET3-19193_combined_Netpilot.actb.bw
bamCoverage -b /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19239_combined_Netpilot.actb.bam -o /project2/gilad/briana/Net-seq-pilot/data/bigwig/YG-SP-NET3-19239_combined_Netpilot.actb.bw
bamCoverage -b /project2/gilad/briana/Net-seq-pilot/data/subset_bam/YG-SP-NET3-19257_combined_Netpilot.actb.bam -o /project2/gilad/briana/Net-seq-pilot/data/bigwig/YG-SP-NET3-19257_combined_Netpilot.actb.bw
```
I also did this for the mayer_hek line.
##Upload to UCSC
Use the instrutions on https://rcc.uchicago.edu/docs/data-transfer/index.html#http-web-access to create a public directory and copy the files to my ~/public_html directory.
'''
http://users.rcc.uchicago.edu/~brimittleman/mayer_hek.actb.bw
http://users.rcc.uchicago.edu/~brimittleman/YG-SP-NET3-19257_combined_Netpilot.actb.bw
http://users.rcc.uchicago.edu/~brimittleman/YG-SP-NET3-19239_combined_Netpilot.actb.bw
http://users.rcc.uchicago.edu/~brimittleman/YG-SP-NET3-19193_combined_Netpilot.actb.bw
http://users.rcc.uchicago.edu/~brimittleman/YG-SP-NET3-19141_combined_Netpilot.actb.bw
http://users.rcc.uchicago.edu/~brimittleman/YG-SP-NET3-18486_combined_Netpilot.actb.bw
http://users.rcc.uchicago.edu/~brimittleman/YG-SP-NET3-18505_combined_Netpilot.actb.bw
http://users.rcc.uchicago.edu/~brimittleman/YG-SP-NET3-18508_combined_Netpilot.actb.bw
http://users.rcc.uchicago.edu/~brimittleman/YG-SP-NET3-19128_combined_Netpilot.actb.bw
'''