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File Version Author Date Message
html c431439 Briana Mittleman 2018-06-13 Build site.
Rmd 772ca2b Briana Mittleman 2018-06-13 picard enrichment plots
html 08b5934 Briana Mittleman 2018-06-13 Build site.
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)
This is workflowr version 1.2.0
Run ?workflowr for help getting started
library(ggplot2)
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(tidyr)
library(reshape2)

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))

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


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] bindrcpp_0.2.2  reshape2_1.4.3  tidyr_0.8.1     dplyr_0.7.6    
[5] ggplot2_3.0.0   workflowr_1.2.0

loaded via a namespace (and not attached):
 [1] Rcpp_0.12.19     compiler_3.5.1   pillar_1.3.0     git2r_0.24.0    
 [5] plyr_1.8.4       bindr_0.1.1      tools_3.5.1      digest_0.6.17   
 [9] evaluate_0.13    tibble_1.4.2     gtable_0.2.0     pkgconfig_2.0.2 
[13] rlang_0.2.2      yaml_2.2.0       withr_2.1.2      stringr_1.4.0   
[17] knitr_1.20       fs_1.2.6         rprojroot_1.3-2  grid_3.5.1      
[21] tidyselect_0.2.4 glue_1.3.0       R6_2.3.0         rmarkdown_1.11  
[25] purrr_0.2.5      magrittr_1.5     whisker_0.3-2    backports_1.1.2 
[29] scales_1.0.0     htmltools_0.3.6  assertthat_0.2.0 colorspace_1.3-2
[33] labeling_0.3     stringi_1.2.4    lazyeval_0.2.1   munsell_0.5.0   
[37] crayon_1.3.4