Last updated: 2018-06-13

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    Rmd 772ca2b Briana Mittleman 2018-06-13 picard enrichment plots
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    Rmd 642faf0 Briana Mittleman 2018-06-13 start PAS enrichment analysis

I am going to use this analysis to look for enrichment of my 3’ seq reads at annoated PAS sites. This is similar to the analysis I ran for the net-seq https://brimittleman.github.io/Net-seq/use_deeptools.html.

Load libraries

library(workflowr)
Loading required package: rmarkdown
This is workflowr version 1.0.1
Run ?workflowr for help getting started
library(ggplot2)
library(dplyr)
Warning: package 'dplyr' was built under R version 3.4.4

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(tidyr)
library(reshape2)
Warning: package 'reshape2' was built under R version 3.4.3

Attaching package: 'reshape2'
The following object is masked from 'package:tidyr':

    smiths

Running Deep Tools:

Step 1: Create bigwig coverage files with bamcoverage

  • bamCoverage -b reads.bam -o coverage.bw

Step 2: computeMatrix

I will need my normalized bigwig reads and the bed interval file (in my case PAS clusters)

ex: computeMatrix scale-regions -S -R -b 1000 -a 1000 -out

–skipZeros (option- not included in first try)

Step 3: Plot heatmap

required –matrixFile, -m (from the compute matrix), -out (file name to save image.png)

–sortRegions descending

–plotTitle, -T

#!/bin/bash


#SBATCH --job-name=deeptools_pas
#SBATCH --time=8:00:00
#SBATCH --partition=broadwl
#SBATCH --mem=40G
#SBATCH --tasks-per-node=4 
#SBATCH --mail-type=END
#SBATCH --output=deeptool_pas_sbatch.out
#SBATCH --error=deeptools_pas_sbatch.err

module load Anaconda3

source activate three-prime-env

sample=$1
describer=$(echo ${sample} | sed -e 's/.*\YL-SP-//' | sed -e "s/-sort.bam$//")


bamCoverage -b $1 -o /project2/gilad/briana/threeprimeseq/output/deeptools/${describer}.bw

computeMatrix reference-point -S project2/gilad/briana/threeprimeseq/output/deeptools/${describer}.bw  -R /project2/gilad/briana/apa_sites/rnaseq_LCL/clusters_fullAnno.bed  -b 500 -a 500 -out /project2/gilad/briana/threeprimeseq/output/deeptools/${describer}.PAS.gz

plotHeatmap --sortRegions descend --refPointLabel "PAS"  -m /project2/gilad/briana/threeprimeseq/output/deeptools/${describer}.PAS.gz  -out /project2/gilad/briana/threeprimeseq/output/deeptools/${describer}.PAS.gz.png

I am running this on YL-SP-18486-N_S10_R1_001-sort.bam to try it first.

Picard statistics

pic.enrich=read.csv("../output/picard/picard.all.enrichment.csv")

pic.enrich.melt=melt(pic.enrich, id.vars="normalized_position") %>% mutate(fraction=ifelse(grepl("T",variable), "total", "nuclear"))%>% mutate(line=substr(variable,3,7))
Warning: package 'bindrcpp' was built under R version 3.4.4

Plot this as line plot:

enrichment.by.line=ggplot(pic.enrich.melt, aes(x=normalized_position, y=value, col=fraction)) + geom_line() + facet_wrap(~line) + labs(y="Normalized Coverage", title="3' Seq enrichment at 3' end of genes", x="Normalized Position") +scale_color_manual(values=c("red", "blue"))
ggsave("../output/plots/enrich.by.line.png", enrichment.by.line)
Saving 7 x 5 in image
enrichment_byfrac=ggplot(pic.enrich.melt, aes(x=normalized_position, y=value, by=line, col=fraction)) + geom_line() + labs(y="Normalized Coverage", title="3' Seq enrichment at 3' end of genes", x="Normalized Position")+ scale_color_manual(values=c("red", "blue"))
ggsave("../output/plots/enrich.by.fraction.png", enrichment_byfrac)
Saving 7 x 5 in image
enrich.by.line.fraction=ggplot(pic.enrich.melt, aes(x=normalized_position, y=value, col=line)) + geom_line() + facet_wrap(~fraction) + labs(y="Normalized Coverage", title="3' Seq enrichment at 3' end of genes", x="Normalized Position") 


ggsave("../output/plots/enrich.by.line.fraction.png",enrich.by.line.fraction)
Saving 7 x 5 in image

Session information

sessionInfo()
R version 3.4.2 (2017-09-28)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS Sierra 10.12.6

Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.4/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  reshape2_1.4.3  tidyr_0.7.2     dplyr_0.7.5    
[5] ggplot2_2.2.1   workflowr_1.0.1 rmarkdown_1.8.5

loaded via a namespace (and not attached):
 [1] Rcpp_0.12.17      compiler_3.4.2    pillar_1.1.0     
 [4] git2r_0.21.0      plyr_1.8.4        bindr_0.1.1      
 [7] R.methodsS3_1.7.1 R.utils_2.6.0     tools_3.4.2      
[10] digest_0.6.15     evaluate_0.10.1   tibble_1.4.2     
[13] gtable_0.2.0      pkgconfig_2.0.1   rlang_0.2.1      
[16] yaml_2.1.19       stringr_1.3.1     knitr_1.18       
[19] rprojroot_1.3-2   grid_3.4.2        tidyselect_0.2.4 
[22] glue_1.2.0        R6_2.2.2          purrr_0.2.5      
[25] magrittr_1.5      whisker_0.3-2     backports_1.1.2  
[28] scales_0.5.0      htmltools_0.3.6   assertthat_0.2.0 
[31] colorspace_1.3-2  labeling_0.3      stringi_1.2.2    
[34] lazyeval_0.2.1    munsell_0.4.3     R.oo_1.22.0

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