Data Processing Figures

Peaks per gene

I want to create a figure similar to the one I created in https://brimittleman.github.io/comparative_threeprime/characterize.ortho.peaks.html. I will use the count distinct function from bedtools map. For this I am using the RefSeq mRNA annotations.

#!/bin/bash

#SBATCH --job-name=refseq_countdistinct
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=refseq_countdistinct.out
#SBATCH --error=refseq_countdistinct.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END



bedtools map -c 4 -s -o count_distinct -a /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.fullchroms.bed -b /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb/filtered_APApeaks_merged_allchrom.named.fixed.bed  > /project2/gilad/briana/threeprimeseq/data/peakPerRefseqGene/filtered_APApeaks_perRefseqGene.txt 

library(tidyverse)

── Attaching packages ──────────────────────────────────────────────────────────────────── tidyverse 1.2.1 ──

✔ ggplot2 3.0.0     ✔ purrr   0.2.5
✔ tibble  1.4.2     ✔ dplyr   0.7.6
✔ tidyr   0.8.1     ✔ stringr 1.3.1
✔ readr   1.1.1     ✔ forcats 0.3.0

── Conflicts ─────────────────────────────────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()

library(workflowr)

This is workflowr version 1.1.1
Run ?workflowr for help getting started

library(reshape2)

Attaching package: 'reshape2'

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

    smiths

library(cowplot)

Attaching package: 'cowplot'

The following object is masked from 'package:ggplot2':

    ggsave

names=c("Chr", "Start", "End", "Name", "Score", "Strand", "numPeaks")
peakpergene=read.table("../data/peakPerRefSeqGene/filtered_APApeaks_perRefseqGene.txt", stringsAsFactors = F, header = F, col.names = names) %>% mutate(onePeak=ifelse(numPeaks==1, 1, 0 )) %>%  mutate(multPeaks=ifelse(numPeaks > 1, 1, 0 ))

genes1peak=sum(peakpergene$onePeak)/nrow(peakpergene) 
genesMultpeak=sum(peakpergene$multPeaks)/nrow(peakpergene)
genes0peak= 1- genes1peak - genesMultpeak

perPeak= c(round(genes0peak,digits = 3), round(genes1peak,digits = 3),round(genesMultpeak, digits = 3))
Category=c("Zero", "One", "Multiple")
perPeakdf=as.data.frame(cbind(Category,as.numeric(perPeak)))

Plot these proportions:

lab1=paste("Genes =", genes0peak*nrow(peakpergene), sep=" ")
lab2=paste("Genes =", sum(peakpergene$onePeak), sep=" ")
lab3=paste("Genes =", sum(peakpergene$multPeaks), sep=" ")

genepeakplot=ggplot(perPeakdf, aes(x="", y=perPeak, fill=Category)) + geom_bar(stat="identity")+ labs(title="Characterize genes by number of PAS", y="Proportion of Protein Coding gene", x="")+ scale_fill_brewer(palette="Paired") + coord_cartesian(ylim=c(0,1)) + annotate("text", x="", y= .35, label=lab1) + annotate("text", x="", y= .78, label=lab2) + annotate("text", x="", y= .92, label=lab3)
genepeakplot

This includes for than 1 isoform for different genes. I am going to go back to the original refseq file and resegment it. Column 13 is the gene name. Column 2 needs to start with NM because that is mRNA.

grep  "NM" ncbiRefSeq.txt | awk '{print $3 "\t" $5 "\t" $6 "\t" $2 "\t" $13 "\t" $4}' > ncbiRefSeq.mRNA.named.bed

I can write a script that writes only the longest isoform for each gene.

outfile=open("refseq.ProteinCoding.bed", "w")




infile=open("ncbiRefSeq.mRNA.named.bed", "r")

lines=infile.readlines()
lot_lines=len(lines)
for n,ln in enumerate(lines):
    chrom, start, end, mRNA, gene, strand = ln.split()
    #if first line
    if n == 0:
        #first line condition
        SE_list=[]
        cur_gene=gene
        SE_list.append(int(start))   
        SE_list.append(int(end)) 
    elif n == lot_lines-1:
        #last line condition
        if gene == cur_gene:
            SE_list.append(int(start))   
            SE_list.append(int(end))
            SE_list.sort()
            outfile.write("%s\t%d\t%d\t%s\t.\t%s\n"%(chrom, SE_list[0], SE_list[-1], gene, strand))
        else: 
           outfile.write("%s\t%d\t%d\t%s\t.\t%s\n"%(chrom, int(start), int(end), gene, strand))
    elif gene == cur_gene:
        SE_list.append(int(start))   
        SE_list.append(int(end))
    elif gene != cur_gene:
        #write out the last line but with the start end from the SE list
        prevline=lines[n-1]
        chrom2, start2, end2, mRNA2, gene2, strand2 = prevline.split()
        outfile.write("%s\t%d\t%d\t%s\t.\t%s\n"%(chrom2, SE_list[0], SE_list[-1], gene2, strand2))
        cur_gene=gene
        SE_list=[int(start), int(end)]


outfile.close()

I can check this by maknig sure there is 1 line for all of the unique names in the in file.

awk '{print $5}' ncbiRefSeq.mRNA.named.bed | sort | uniq | wc -l
#19243
wc -l refseq.ProteinCoding.bed 
#20298
sed 's/^chr//' refseq.ProteinCoding.bed > refseq.ProteinCoding.noCHR.bed

There is still a problem with the script. The problem is when the same gene name is on extra haplotypes. I could remove all of the lines in the file that have _ in the first column. These are on contigs or specfic haplotypes. They will not map to our peaks anyway. I also need to remove the chr.

This still seems lower than previos APA estimates. I had used gencode estimates before. I am gonig to run this analysis again with those gene.

#!/bin/bash

#SBATCH --job-name=gencode_countdistinct
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=gencode_countdistinct.out
#SBATCH --error=gencode_countdistinct.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END



bedtools map -c 4 -s -o count_distinct -a /project2/gilad/briana/genome_anotation_data/gencode.v19.annotation.proteincodinggene.sort.bed   -b /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb/filtered_APApeaks_merged_allchrom.named.fixed.bed  >

Gpeakpergene=read.table("../data/peakPerRefSeqGene/filtered_APApeaks_perGencodeGene.txt", stringsAsFactors = F, header = F, col.names = names) %>% mutate(onePeak=ifelse(numPeaks==1, 1, 0 )) %>%  mutate(multPeaks=ifelse(numPeaks > 1, 1, 0 ))

Ggenes1peak=sum(Gpeakpergene$onePeak)/nrow(Gpeakpergene) 
GgenesMultpeak=sum(Gpeakpergene$multPeaks)/nrow(Gpeakpergene)
Ggenes0peak= 1- Ggenes1peak - GgenesMultpeak

GperPeak= c(round(Ggenes0peak,digits = 3), round(Ggenes1peak,digits = 3),round(GgenesMultpeak, digits = 3))

GperPeakdf=as.data.frame(cbind(Category,as.numeric(GperPeak)))

Plot these proportions:

Glab1=paste("Genes =", Ggenes0peak*nrow(Gpeakpergene), sep=" ")
Glab2=paste("Genes =", sum(Gpeakpergene$onePeak), sep=" ")
Glab3=paste("Genes =", sum(Gpeakpergene$multPeaks), sep=" ")

Ggenepeakplot=ggplot(GperPeakdf, aes(x="", y=perPeak, fill=Category)) + geom_bar(stat="identity")+ labs(title="Characterize Gencode genes by number of PAS", y="Proportion of Protein Coding gene", x="")+ scale_fill_brewer(palette="Paired") + coord_cartesian(ylim=c(0,1)) + annotate("text", x="", y= .35, label=Glab1) + annotate("text", x="", y= .78, label=Glab2) + annotate("text", x="", y= .92, label=Glab3)
Ggenepeakplot

These results are still lower than expected. This is because all of my previous analysis mapped the genes to the peaks as which were closest in the upstream direction. Here I am saying the peak must overlap the gene.

Session information

sessionInfo()

R version 3.5.1 (2018-07-02)
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/3.5/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  cowplot_0.9.3   reshape2_1.4.3  workflowr_1.1.1
 [5] forcats_0.3.0   stringr_1.3.1   dplyr_0.7.6     purrr_0.2.5    
 [9] readr_1.1.1     tidyr_0.8.1     tibble_1.4.2    ggplot2_3.0.0  
[13] tidyverse_1.2.1

loaded via a namespace (and not attached):
 [1] tidyselect_0.2.4   haven_1.1.2        lattice_0.20-35   
 [4] colorspace_1.3-2   htmltools_0.3.6    yaml_2.2.0        
 [7] rlang_0.2.2        R.oo_1.22.0        pillar_1.3.0      
[10] glue_1.3.0         withr_2.1.2        R.utils_2.7.0     
[13] RColorBrewer_1.1-2 modelr_0.1.2       readxl_1.1.0      
[16] bindr_0.1.1        plyr_1.8.4         munsell_0.5.0     
[19] gtable_0.2.0       cellranger_1.1.0   rvest_0.3.2       
[22] R.methodsS3_1.7.1  evaluate_0.11      labeling_0.3      
[25] knitr_1.20         broom_0.5.0        Rcpp_0.12.18      
[28] scales_1.0.0       backports_1.1.2    jsonlite_1.5      
[31] hms_0.4.2          digest_0.6.16      stringi_1.2.4     
[34] grid_3.5.1         rprojroot_1.3-2    cli_1.0.0         
[37] tools_3.5.1        magrittr_1.5       lazyeval_0.2.1    
[40] crayon_1.3.4       whisker_0.3-2      pkgconfig_2.0.2   
[43] xml2_1.2.0         lubridate_1.7.4    assertthat_0.2.0  
[46] rmarkdown_1.10     httr_1.3.1         rstudioapi_0.7    
[49] R6_2.2.2           nlme_3.1-137       git2r_0.23.0      
[52] compiler_3.5.1