Last updated: 2018-12-13

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Unstaged changes:
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    Modified:   analysis/dif.iso.usage.leafcutter.Rmd
    Modified:   analysis/diff_iso_pipeline.Rmd
    Modified:   analysis/explainpQTLs.Rmd
    Modified:   analysis/explore.filters.Rmd
    Modified:   analysis/flash2mash.Rmd
    Modified:   analysis/overlapMolQTL.Rmd
    Modified:   analysis/overlap_qtls.Rmd
    Modified:   analysis/peakOverlap_oppstrand.Rmd
    Modified:   analysis/pheno.leaf.comb.Rmd
    Modified:   analysis/swarmPlots_QTLs.Rmd
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    File Version Author Date Message
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    Rmd b198e3b Briana Mittleman 2018-12-13 4kb around end
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    Rmd 23123cf Briana Mittleman 2018-12-12 add only genes not nearby
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    Rmd 0e0840e Briana Mittleman 2018-12-12 remove overlaping genes
    html 6da90e9 Briana Mittleman 2018-12-11 Build site. 

library(tidyverse)
── Attaching packages ───────────────────────────────────────────────────────── tidyverse 1.2.1 ──
✔ ggplot2 3.0.0     ✔ purrr   0.2.5
✔ tibble  1.4.2     ✔ dplyr   0.7.6
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── Conflicts ──────────────────────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
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library(workflowr)
This is workflowr version 1.1.1
Run ?workflowr for help getting started
library(cowplot)

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

    ggsave
library(tximport)

In looking at correlations and some examples, there is evidence the peak to gene assignment may be a problem. I am going to visualize the peaks in IGV. I will name them by the gene and look at them in the browser.

The peak to gene annotations used in the feature counts to map reads back to the peaks is the following:
* /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed.bed

I need to change this a bit to have the name be the gene rather than the score:

NamePeaksByGene.py

#python  

CovnamedPeaks=open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed.bed", "r")
GeneNamedPeaks=open("/project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/PeaksNamedWithGeneAssignment.bed", "w")

for ln in CovnamedPeaks:
  chrom, start, end, num, cov, strand, transcript = ln.split()
  gene=transcript.split("-")[1]
  GeneNamedPeaks.write("%s\t%s\t%s\t%s\n"%(chrom,start,end,gene))

GeneNamedPeaks.close()

This was made based on the transcript annotation: ncbiRefSeq.mRNA.named.bed

  • /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named.bed

The ends of the transcripts specfically are in:

  • /project2/gilad/briana/genome_anotation_data/ncbiRefSeq_endProtCodGenes_sort.txt

Ideas for Dilters:

  • Cant be upstream of the gene, ex: chr2:135,558,075-135,604,343

  • maybe it cant be in another gene

  • we should include LINCs

  • looks like we have a ton of low expressed intergenic peaks that should be filtered before we do the gene annotation

Filter out intergenic peaks

As a first pass I want to filter out the peaks that are outside a gene body. While this may not be perfect it will help alot with the intergenic noise.

I need to overlap the named peaks with /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named.bed and only keep the matches. I can use bedtools intersect.

Rename the peaks according to convention to run an intesect.

RenamePeaks4Intersect.py

#python  
CovnamedPeaks=open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed.bed", "r")
GeneNamedPeaks=open("/project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED.bed", "w")  

for ln in CovnamedPeaks:
  chrom, start, end, num, cov, strand, transcript = ln.split()
  gene=transcript.split("-")[1]
  start=int(start)
  end=int(end)
  GeneNamedPeaks.write("%s\t%d\t%d\t%s-%s\t%s\t%s\n"%(chrom,start,end,num,gene,cov,strand))

GeneNamedPeaks.close()

Remove CHR from the refseq annpotation:

sed 's/^chr//' /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named.bed > /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR.bed

Filter4GenicPeaks.sh


#!/bin/bash


#SBATCH --job-name=Filter4GenicPeaks
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=Filter4GenicPeaks.out
#SBATCH --error=Filter4GenicPeaks.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


bedtools intersect -wa -s -a /project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED.bed -b /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR.bed > /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies.bed

This is printing them multiple times.

uniq /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies.bed > /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodiesUNIQ.bed

Now I need to make this an SAF to run feature counts.

bed2saf_peaksInGenicReg.py

from misc_helper import *

fout = open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodiesUNIQ.SAF",'w')
fout.write("GeneID\tChr\tStart\tEnd\tStrand\n")
for ln in open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodiesUNIQ.bed"):
    chrom, start, end, name, score, strand = ln.split()
    namenum=name.split("-")[0]
    name_i=int(namenum)
    start_i=int(start)
    end_i=int(end)
    gene_only=name.split("-")[1]
    ID = "peak%d:%s:%d:%d:%s:%s"%(name_i, chrom, start_i, end_i, strand, gene_only)
    fout.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
fout.close()

Run Feature Counts
PeaksinGenicRegion_fc_TN.sh

#!/bin/bash

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

module load Anaconda3
source activate three-prime-env

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodiesUNIQ.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/PeakInGenecRegion_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Genic.Total.fc /project2/gilad/briana/threeprimeseq/data/sort/*-T-*-sort.bam -s 2

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodiesUNIQ.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/PeakInGenecRegion_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Genic.Nuclear.fc /project2/gilad/briana/threeprimeseq/data/sort/*-N-*-sort.bam -s 2

Lastly I will need to fix the headers.

fix_head_fc_genicPeak_tot.py

infile= open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegion_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Genic.Total.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegion_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Genic.Total_fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

fix_head_fc_genicPeak_nuc.py

infile= open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegion_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Genic.Nuclear.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegion_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Genic.Nuclear_fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

Pull these into R and look at the correlation between the sum of the peaks by gene and the transcripts counts from RNA seq.

TPM counts from Kalisto

tx2gene=read.table("../data/RNAkalisto/ncbiRefSeq.txn2gene.txt" ,header= F, sep="\t", stringsAsFactors = F)

txi.kallisto.tsv <- tximport("../data/RNAkalisto/abundance.tsv", type = "kallisto", tx2gene = tx2gene,countsFromAbundance="lengthScaledTPM" )
Note: importing `abundance.h5` is typically faster than `abundance.tsv`
reading in files with read_tsv
1 
removing duplicated transcript rows from tx2gene
transcripts missing from tx2gene: 99
summarizing abundance
summarizing counts
summarizing length

In previous analysis I did not account for gene length. Here I am going to standardize by length because I am taking a sum over a gene body.

Import gene lengths:

geneLengthNames=c("CHR", "start", "end", "gene", "score", "strand")
geneLengths=read.table("../data/UnderstandPeaksQC/refseq.ProteinCoding.bed", header=F, stringsAsFactors = F, col.names = geneLengthNames) %>% mutate(length=end-start) %>% select(gene, length)

Look at the correlation with the total:

I am using the sum of the counts in a gene divided by how many million reads mapped. I am also filtering out peaks with less than 10 reads in this individual.

total_Cov_18486=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Genic.Total_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_T) %>% filter(X18486_T>0) %>%  group_by(gene) %>% summarize(GeneSum=sum(X18486_T)) %>% mutate(GeneSumNorm=GeneSum/10.8) %>% inner_join(geneLengths, by="gene") %>% mutate(GeneSumSt=GeneSum/length)

Join the data frames.

TXN_abund=as.data.frame(txi.kallisto.tsv$abundance) %>% rownames_to_column(var="gene")
colnames(TXN_abund)=c("gene", "TPM")

TXN_NormGene=TXN_abund %>% inner_join(total_Cov_18486,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene=TXN_NormGene %>% filter(TPM>0) %>% filter(GeneSumSt>0)
corr_18486Tot=ggplot(TXN_NormGene, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Total", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.48") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') 

#+ geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Tot

Expand here to see past versions of unnamed-chunk-15-1.png:
Version Author Date
558e8e8 Briana Mittleman 2018-12-12 

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.5894 -0.2556  0.0856  0.3676  2.3387 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)      2.407969   0.013563   177.5   <2e-16 ***
log10(GeneSumSt) 0.612175   0.005812   105.3   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.598 on 12053 degrees of freedom
Multiple R-squared:  0.4793,    Adjusted R-squared:  0.4793 
F-statistic: 1.11e+04 on 1 and 12053 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene$TPM),log10(TXN_NormGene$GeneSumSt))

    Pearson's product-moment correlation

data:  log10(TXN_NormGene$TPM) and log10(TXN_NormGene$GeneSumSt)
t = 105.34, df = 12053, p-value < 2.2e-16
alternative hypothesis: true correlation is not equal to 0
95 percent confidence interval:
 0.6829262 0.7015184
sample estimates:
      cor 
0.6923372

Try this with nuclear

nuclear_Cov_18486=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Genic.Nuclear_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_N) %>% filter(X18486_N>10) %>%  group_by(gene) %>% summarize(GeneSum=sum(X18486_N)) %>% mutate(GeneSumNorm=GeneSum/11.4) %>% inner_join(geneLengths, by="gene") %>% mutate(GeneSumSt=GeneSum/length)

Join the data frames.

TXN_NormGene_nuc=TXN_abund %>% inner_join(nuclear_Cov_18486,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene_nuc=TXN_NormGene_nuc %>% filter(TPM>0) %>% filter(GeneSumSt>0)
corr_18486Nuc=ggplot(TXN_NormGene_nuc, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Nuclear", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.37") + geom_density2d(na.rm = TRUE, size = 1, colour = 'red') 

#+ geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Nuc

Expand here to see past versions of unnamed-chunk-18-1.png:
Version Author Date
558e8e8 Briana Mittleman 2018-12-12 

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene_nuc)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene_nuc)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.7211 -0.2691  0.0733  0.3789  2.5253 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)      2.451150   0.017039  143.85   <2e-16 ***
log10(GeneSumSt) 0.654587   0.008008   81.74   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.6324 on 11567 degrees of freedom
Multiple R-squared:  0.3661,    Adjusted R-squared:  0.3661 
F-statistic:  6681 on 1 and 11567 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_nuc$TPM),log10(TXN_NormGene_nuc$GeneSumSt))

    Pearson's product-moment correlation

data:  log10(TXN_NormGene_nuc$TPM) and log10(TXN_NormGene_nuc$GeneSumSt)
t = 81.74, df = 11567, p-value < 2.2e-16
alternative hypothesis: true correlation is not equal to 0
95 percent confidence interval:
 0.593414 0.616518
sample estimates:
      cor 
0.6050934

This just said it had to be in a gene body not the specific gene body. This could be a problem still. For example in the SSPO locus chr7:149,521,993-149,543,749. Here the peaks are closer to the end of the SSPO but are in the gene body of the next gene downstream.

Histones dont have a polyA tail- the HIST1H4C peak is most likely misprimming (chr6:26,102,306-26,110,443)

Filter out overlapping genes:

Count overlaps in origial file:
bedtools merge -i IN.bed -c 1 -o count > counted

countGeneOverlap.sh

#!/bin/bash

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

module load Anaconda3
source activate three-prime-env


bedtools merge -i /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.noCHR.bed -c 1 -o count > /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.countGeneOverlap.bed

Filter out these rows: awk '/\t1$/{print}' counted > filtered

awk '/\t1$/{print}' /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.countGeneOverlap.bed > /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.countGeneOverlap.filtered.bed

Intersect with original input to only keep the ones in both sets.
bedtools intersect -a IN.bed -b filtered -wa > OUT.bed

findGeneswithoutOverlap.sh

#!/bin/bash

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

module load Anaconda3
source activate three-prime-env

bedtools intersect -a /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.noCHR.bed -b /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.countGeneOverlap.filtered.bed -wa > /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.NonOverlapGenes.bed

Finally overlap with the mRNA file to only keep the transcripts in these genes. This may be easiest in python /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR.bed

subsetmRNAforNonOverlapGenes.py

geneFile=open("/project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.NonOverlapGenes.bed", "r") 

mRNAFile=open("/project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR.bed", "r")

outFile=open("/project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR_NoneOverlapingGenes.bed", "w")  

#make list of non overlapping genes  

keep=[]
for ln in geneFile:
  keep.append(ln.split()[3])

for ln in mRNAFile: 
  if ln.split()[4] in keep:
    outFile.write(ln)
outFile.close()

Filter peaks on this resutls

Filter4GenicPeaks_noOverlap.sh


#!/bin/bash


#SBATCH --job-name=Filter4GenicPeaks
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=Filter4GenicPeaks.out
#SBATCH --error=Filter4GenicPeaks.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


bedtools intersect -wa -s -a /project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED.bed -b /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR_NoneOverlapingGenes.bed> /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlap.bed

This is printing them multiple times.

uniq /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlap.bed > /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlap_UNIQ.bed

Make this an SAF to run FC

bed2saf_peaksInGenicReg_noOVERLAP.py

from misc_helper import *

fout = open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlap_UNIQ.SAF",'w')
fout.write("GeneID\tChr\tStart\tEnd\tStrand\n")
for ln in open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlap_UNIQ.bed"):
    chrom, start, end, name, score, strand = ln.split()
    namenum=name.split("-")[0]
    name_i=int(namenum)
    start_i=int(start)
    end_i=int(end)
    gene_only=name.split("-")[1]
    ID = "peak%d:%s:%d:%d:%s:%s"%(name_i, chrom, start_i, end_i, strand, gene_only)
    fout.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
fout.close()

Run Feature Counts
PeaksinGenicRegion_NoneOverlapGenes_fc_TN.sh

#!/bin/bash

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

module load Anaconda3
source activate three-prime-env

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlap_UNIQ.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlap_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Total.fc /project2/gilad/briana/threeprimeseq/data/sort/*-T-*-sort.bam -s 2

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlap_UNIQ.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlap_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Nuclear.fc /project2/gilad/briana/threeprimeseq/data/sort/*-N-*-sort.bam -s 2

Lastly I will need to fix the headers.

fix_head_fc_genicPeakNoOverlap_tot.py

infile= open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlap_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Total.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlap_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Total_fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

fix_head_fc_genicPeakNoOverlap_nuc.py

infile= open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlap_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Nuclear.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlap_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Nuclear_fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

Pull these onto my computer:

Use no filter and standardization scheme:

total_Cov_18486_noOver=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Total_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_T)%>%  group_by(gene) %>% summarize(GeneSum=sum(X18486_T)) %>% mutate(GeneSumNorm=GeneSum/10.8) %>% inner_join(geneLengths, by="gene") %>% mutate(GeneSumSt=GeneSum/length)

Join the data frames.

TXN_NormGene_noOverlap=TXN_abund %>% inner_join(total_Cov_18486_noOver,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene_noOverlap=TXN_NormGene_noOverlap %>% filter(TPM>0) %>% filter(GeneSumSt>0)
corr_18486Tot_noOver=ggplot(TXN_NormGene_noOverlap, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Total", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.49") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') + geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Tot_noOver

Expand here to see past versions of unnamed-chunk-31-1.png:
Version Author Date
cbf986c Briana Mittleman 2018-12-12 

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene_noOverlap)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene_noOverlap)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.3953 -0.2604  0.0768  0.3638  2.8920 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)      2.435113   0.014849  163.99   <2e-16 ***
log10(GeneSumSt) 0.616937   0.006274   98.34   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.5973 on 10088 degrees of freedom
Multiple R-squared:  0.4894,    Adjusted R-squared:  0.4894 
F-statistic:  9671 on 1 and 10088 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlap$TPM),log10(TXN_NormGene_noOverlap$GeneSumSt))

    Pearson's product-moment correlation

data:  log10(TXN_NormGene_noOverlap$TPM) and log10(TXN_NormGene_noOverlap$GeneSumSt)
t = 98.339, df = 10088, p-value < 2.2e-16
alternative hypothesis: true correlation is not equal to 0
95 percent confidence interval:
 0.6894966 0.7094251
sample estimates:
      cor 
0.6995969 
nuclear_Cov_18486_noOver=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Nuclear_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_N) %>%  group_by(gene) %>% summarize(GeneSum=sum(X18486_N)) %>% mutate(GeneSumNorm=GeneSum/11.4) %>% inner_join(geneLengths, by="gene") %>% mutate(GeneSumSt=GeneSum/length)

Join the data frames.

TXN_NormGene_noOverlap_nuc=TXN_abund %>% inner_join(nuclear_Cov_18486_noOver,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene_noOverlap_nuc=TXN_NormGene_noOverlap_nuc %>% filter(TPM>0) %>% filter(GeneSumSt>0)
corr_18486Nuc_noOver=ggplot(TXN_NormGene_noOverlap_nuc, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Nuclear", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.5") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') 

#+ geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Nuc_noOver

Expand here to see past versions of unnamed-chunk-34-1.png:
Version Author Date
cbf986c Briana Mittleman 2018-12-12 

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene_noOverlap_nuc)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene_noOverlap_nuc)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.6947 -0.2989  0.0630  0.3825  2.8300 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)      2.530468   0.016063   157.5   <2e-16 ***
log10(GeneSumSt) 0.708816   0.006819   103.9   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.647 on 10965 degrees of freedom
Multiple R-squared:  0.4963,    Adjusted R-squared:  0.4963 
F-statistic: 1.08e+04 on 1 and 10965 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlap_nuc$TPM),log10(TXN_NormGene_noOverlap_nuc$GeneSumSt),method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_noOverlap_nuc$TPM),
log10(TXN_NormGene_noOverlap_nuc$GeneSumSt), : Cannot compute exact p-value
with ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_noOverlap_nuc$TPM) and log10(TXN_NormGene_noOverlap_nuc$GeneSumSt)
S = 6.8486e+10, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.6884764

It looks like a more stringent RNA seq filter could help. Lets say log(TPM)>-1.5

TXN_abund_filt=TXN_abund %>% filter(log(TPM) > -1.5)
TXN_NormGene_noOverlap_filt=TXN_abund_filt %>% inner_join(total_Cov_18486_noOver,by="gene")
TXN_NormGene_noOverlap_filt=TXN_NormGene_noOverlap_filt %>% filter(TPM >0) %>% filter(GeneSumSt>0)
corr_18486Tot_noOver_filt=ggplot(TXN_NormGene_noOverlap_filt, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Total filtered", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.49") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') 

#+ geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Tot_noOver_filt

Expand here to see past versions of unnamed-chunk-35-1.png:
Version Author Date
cbf986c Briana Mittleman 2018-12-12 

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene_noOverlap_filt)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene_noOverlap_filt)

Residuals:
     Min       1Q   Median       3Q      Max 
-3.05828 -0.26269  0.03857  0.31263  2.64091 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)      2.344999   0.012950  181.08   <2e-16 ***
log10(GeneSumSt) 0.544908   0.005639   96.63   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.4998 on 9677 degrees of freedom
Multiple R-squared:  0.4911,    Adjusted R-squared:  0.491 
F-statistic:  9338 on 1 and 9677 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlap_filt$TPM),log10(TXN_NormGene_noOverlap_filt$GeneSumSt),method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_noOverlap_filt$TPM),
log10(TXN_NormGene_noOverlap_filt$GeneSumSt), : Cannot compute exact p-
value with ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_noOverlap_filt$TPM) and log10(TXN_NormGene_noOverlap_filt$GeneSumSt)
S = 4.7005e+10, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.6889706

This does not take care of genes that are near each other such as what is going on with SSPO

Filter genes that are too close together

This is similar to the overlap problem but I want to extend the genes.

I can a python script to subtract 10000 bases from the start and add 10000 to the end

*/project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.noCHR.bed

extendGenes.py

inFile=open("/project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.noCHR.bed", "r")

outFile=open("/project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.noCHR_EXT.bed", "w")\


for ln in inFile:
  chrom, start, end, gene, score, strand = ln.split()
  start_ex=int(start)-10000
  if start_ex <0: 
    start_ex=0
  end_ex=int(end)+10000
  outFile.write("%s\t%d\t%d\t%s\t%s\t%s\n"%(chrom, start_ex, end_ex, gene, score, strand))
  
outFile.close()

Now I can run the merge:

countGeneOverlap_EXT.sh

#!/bin/bash

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

module load Anaconda3
source activate three-prime-env


bedtools merge -i /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.noCHR_EXT.bed -c 1 -o count > /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.countGeneOverlap_EXT.bed

Filter out these rows: awk '/\t1$/{print}' counted > filtered

awk '/\t1$/{print}' /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.countGeneOverlap_EXT.bed > /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.countGeneOverlap_EXT_filter.bed

Intersect with original input to only keep the ones in both sets.
bedtools intersect -a IN.bed -b filtered -wa > OUT.bed

findGeneswithoutOverlap_EXT.sh

#!/bin/bash

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

module load Anaconda3
source activate three-prime-env

bedtools intersect -a /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.noCHR.bed -b /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.countGeneOverlap_EXT_filter.bed -wa > /project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.NonOverlapGenes_EXT.bed

Finally overlap with the mRNA file to only keep the transcripts in these genes. This may be easiest in python /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR.bed

subsetmRNAforNonOverlapGenes_EXT.py

geneFile=open("/project2/gilad/briana/genome_anotation_data/refseq.ProteinCoding.NonOverlapGenes_EXT.bed", "r") 

mRNAFile=open("/project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR.bed", "r")

outFile=open("/project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR_NoneOverlapingGenes_EXT.bed", "w")  

#make list of non overlapping genes  

keep=[]
for ln in geneFile:
  keep.append(ln.split()[3])

for ln in mRNAFile: 
  if ln.split()[4] in keep:
    outFile.write(ln)
outFile.close()

Filter4GenicPeaks_noOverlap_EXT.sh


#!/bin/bash


#SBATCH --job-name=Filter4GenicPeaks_EXT
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=Filter4GenicPeaks_EXT.out
#SBATCH --error=Filter4GenicPeaks_EXT.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


bedtools intersect -wa -s -a /project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED.bed -b /project2/gilad/briana/genome_anotation_data/ncbiRefSeq.mRNA.named_noCHR_NoneOverlapingGenes_EXT.bed > /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlapEXT.bed

This is printing them multiple times.

uniq /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlapEXT.bed > /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlapEXT_UNIQ.bed

Make this an SAF to run FC

bed2saf_peaksInGenicReg_noOVERLAPEXT.py

from misc_helper import *

fout = open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlapEXT_UNIQ.SAF",'w')
fout.write("GeneID\tChr\tStart\tEnd\tStrand\n")
for ln in open("/project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlapEXT_UNIQ.bed"):
    chrom, start, end, name, score, strand = ln.split()
    namenum=name.split("-")[0]
    name_i=int(namenum)
    start_i=int(start)
    end_i=int(end)
    gene_only=name.split("-")[1]
    ID = "peak%d:%s:%d:%d:%s:%s"%(name_i, chrom, start_i, end_i, strand, gene_only)
    fout.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
fout.close()

Run Feature Counts
PeaksinGenicRegion_NoneOverlapGenesEXT_fc_TN.sh

#!/bin/bash

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

module load Anaconda3
source activate three-prime-env

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlapEXT_UNIQ.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlapEXT_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total.fc /project2/gilad/briana/threeprimeseq/data/sort/*-T-*-sort.bam -s 2

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb_inGeneBody/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_inGeneBodies_noGeneOverlapEXT_UNIQ.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlapEXT_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Nuclear.fc /project2/gilad/briana/threeprimeseq/data/sort/*-N-*-sort.bam -s 2

Lastly I will need to fix the headers.

fix_head_fc_genicPeakNoOverlapEXT_tot.py

infile= open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlapEXT_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlapEXT_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total_fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

fix_head_fc_genicPeakNoOverlapEXT_nuc.py

infile= open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlapEXT_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Nuclear.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/PeakInGenecRegionNoOverlapEXT_cov/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Nuclear_Fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

Pull these onto my computer:

Use no filter and standardization scheme:

total_Cov_18486_noOverEXT=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_T)%>%  group_by(gene) %>% summarize(GeneSum=sum(X18486_T)) %>% mutate(GeneSumNorm=GeneSum/10.8)

Join the data frames.

TXN_NormGene_noOverlapEXT=TXN_abund %>% inner_join(total_Cov_18486_noOverEXT,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene_noOverlapEXT_n=TXN_NormGene_noOverlapEXT %>% filter(TPM>0) %>% filter(GeneSumNorm>0)
corr_18486Tot_noOverEXT=ggplot(TXN_NormGene_noOverlapEXT_n, aes(x=log10(TPM), y= log10(GeneSumNorm))) + geom_point() + labs(title="Total", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumNorm)),method = "lm") + annotate("text",x=3, y=5,label="R2=.42") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red')

#+geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Tot_noOverEXT

Expand here to see past versions of unnamed-chunk-49-1.png:
Version Author Date
c3fd2c4 Briana Mittleman 2018-12-13
cbf986c Briana Mittleman 2018-12-12 

summary(lm(log10(TPM)~log10(GeneSumNorm),TXN_NormGene_noOverlapEXT_n)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumNorm), data = TXN_NormGene_noOverlapEXT_n)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.6242 -0.2842  0.0307  0.3445  2.9033 

Coefficients:
                   Estimate Std. Error t value Pr(>|t|)    
(Intercept)         0.18874    0.02195   8.598   <2e-16 ***
log10(GeneSumNorm)  0.66519    0.01540  43.200   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.6428 on 2542 degrees of freedom
Multiple R-squared:  0.4234,    Adjusted R-squared:  0.4231 
F-statistic:  1866 on 1 and 2542 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlapEXT_n$TPM),log10(TXN_NormGene_noOverlapEXT_n$GeneSumNorm),method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_noOverlapEXT_n$TPM),
log10(TXN_NormGene_noOverlapEXT_n$GeneSumNorm), : Cannot compute exact p-
value with ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_noOverlapEXT_n$TPM) and log10(TXN_NormGene_noOverlapEXT_n$GeneSumNorm)
S = 880650000, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.6790751

In this test, I was looking at 2600 genes.

nuclear_Cov_18486_noOverEXT=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Nuclear_Fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_N) %>%  group_by(gene) %>% summarize(GeneSum=sum(X18486_N)) %>% mutate(GeneSumNorm=GeneSum/11.4)

Join the data frames.

TXN_NormGene_noOverlap_nucEXT=TXN_abund %>% inner_join(nuclear_Cov_18486_noOverEXT,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene_noOverlap_nucEXT_n=TXN_NormGene_noOverlap_nucEXT %>% filter(TPM>0) %>% filter(GeneSumNorm>0)
corr_18486Nuc_noOverEXT=ggplot(TXN_NormGene_noOverlap_nucEXT_n, aes(x=log10(TPM), y= log10(GeneSumNorm))) + geom_point() + labs(title="Nuclear", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumNorm)),method = "lm") + annotate("text",x=3, y=5,label="R2=.43") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') 

#+geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Nuc_noOverEXT

Expand here to see past versions of unnamed-chunk-52-1.png:
Version Author Date
c3fd2c4 Briana Mittleman 2018-12-13
cbf986c Briana Mittleman 2018-12-12 

summary(lm(log10(TPM)~log10(GeneSumNorm),TXN_NormGene_noOverlap_nucEXT_n)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumNorm), data = TXN_NormGene_noOverlap_nucEXT_n)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.4278 -0.3689  0.0112  0.3873  3.2887 

Coefficients:
                   Estimate Std. Error t value Pr(>|t|)    
(Intercept)         0.02478    0.02179   1.137    0.256    
log10(GeneSumNorm)  0.68406    0.01470  46.527   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.7076 on 2836 degrees of freedom
Multiple R-squared:  0.4329,    Adjusted R-squared:  0.4327 
F-statistic:  2165 on 1 and 2836 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlap_nucEXT_n$TPM),log10(TXN_NormGene_noOverlap_nucEXT_n$GeneSumNorm),method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_noOverlap_nucEXT_n$TPM), :
Cannot compute exact p-value with ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_noOverlap_nucEXT_n$TPM) and log10(TXN_NormGene_noOverlap_nucEXT_n$GeneSumNorm)
S = 1351600000, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.6452286

In this test, I was looking at 2900 genes.

I can further ssubset for genes with only 1 peak.

OnePeakGenesTotalnoOver=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_T)%>%  group_by(gene) %>% tally() %>% filter(n>=3)

total_Cov_18486_noOverEXT_1Peak=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_T)%>%  group_by(gene) %>% summarize(GeneSum=sum(X18486_T)) %>% mutate(GeneSumNorm=GeneSum/10.8) %>% inner_join(geneLengths, by="gene") %>% mutate(GeneSumSt=GeneSum/length) %>% semi_join(OnePeakGenesTotalnoOver, by="gene")
TXN_NormGene_noOverlapEXT_1peak=TXN_abund %>% inner_join(total_Cov_18486_noOverEXT_1Peak,by="gene")
TXN_NormGene_noOverlapEXT_1peak_n=TXN_NormGene_noOverlapEXT_1peak %>% filter(TPM>0) %>% filter(GeneSumNorm>0)
corr_18486Tot_noOverEXT=ggplot(TXN_NormGene_noOverlapEXT_1peak_n, aes(x=log10(TPM), y= log10(GeneSumNorm))) + geom_point() + labs(title="Total-nonOverlapping Genes with 3 peaks", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumNorm)),method = "lm") + annotate("text",x=3, y=5,label="R2=.4") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') 

#+geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Tot_noOverEXT

Expand here to see past versions of unnamed-chunk-55-1.png:
Version Author Date
cbf986c Briana Mittleman 2018-12-12 

summary(lm(log10(TPM)~log10(GeneSumNorm),TXN_NormGene_noOverlapEXT_1peak_n)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumNorm), data = TXN_NormGene_noOverlapEXT_1peak_n)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.2185 -0.2785  0.0258  0.3296  2.7657 

Coefficients:
                   Estimate Std. Error t value Pr(>|t|)    
(Intercept)         0.27643    0.02297   12.03   <2e-16 ***
log10(GeneSumNorm)  0.61688    0.01570   39.30   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.5918 on 2366 degrees of freedom
Multiple R-squared:  0.395, Adjusted R-squared:  0.3947 
F-statistic:  1544 on 1 and 2366 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlapEXT_1peak_n$TPM),log10(TXN_NormGene_noOverlapEXT_1peak_n$GeneSumNorm),method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_noOverlapEXT_1peak_n$TPM), :
Cannot compute exact p-value with ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_noOverlapEXT_1peak_n$TPM) and log10(TXN_NormGene_noOverlapEXT_1peak_n$GeneSumNorm)
S = 752880000, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.6598005

Around gene end

There are still problems with peaks in the downstream gene. I need to have a filter that a peak needs to be within a certain distance from the end of a gene. I can change the end of gene file to have 2kb around each gene end and then work with peaks in this area. I want it to be 2kb into the gene.

I can filter the peaks to only those in these regions.

EndOfGenes_4kbaround.py

#python  

inFile=open("/project2/gilad/briana/genome_anotation_data/ncbiRefSeq_endProtCodGenes_sort_withscore.bed", "r")

outFile=open("/project2/gilad/briana/genome_anotation_data/ncbiRefSeq_4KBaroundEnd_sort_withscore.bed", "w")\


for ln in inFile:
  chrom, start, end, gene, score, strand = ln.split()
  start_ex=int(start)-2000
  if start_ex <0: 
    start_ex=0
  end_ex=int(end)+2000
  outFile.write("%s\t%d\t%d\t%s\t%s\t%s\n"%(chrom, start_ex, end_ex, gene, score, strand))
  
outFile.close()

Now I intersect this with the peaks file.

FilterPeaks4KBaroundend.sh

#!/bin/bash


#SBATCH --job-name=FilterPeaks4KBaroundend
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=FilterPeaks4KBaroundend.out
#SBATCH --error=FilterPeaks4KBaroundend.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env


bedtools intersect -a /project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED.bed -b /project2/gilad/briana/genome_anotation_data/ncbiRefSeq_4KBaroundEnd_sort_withscore.bed -wa -s  > /project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED_4kbaroundEnd.bed

This is printing them multiple times.

uniq /project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED_4kbaroundEnd.bed > /project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED_4kbaroundEnd.UNIQ.bed

Make this an SAF to run FC

bed2saf_peaksInGenicReg_4kbaround.py

from misc_helper import *

fout = open("/project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED_4kbaroundEnd.UNIQ.SAF",'w')
fout.write("GeneID\tChr\tStart\tEnd\tStrand\n")
for ln in open("/project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED_4kbaroundEnd.UNIQ.bed"):
    chrom, start, end, name, score, strand = ln.split()
    namenum=name.split("-")[0]
    name_i=int(namenum)
    start_i=int(start)
    end_i=int(end)
    gene_only=name.split("-")[1]
    ID = "peak%d:%s:%d:%d:%s:%s"%(name_i, chrom, start_i, end_i, strand, gene_only)
    fout.write("%s\t%s\t%d\t%d\t%s\n"%(ID, chrom, start_i, end_i, strand))
fout.close()

Run feature counts with this.
Peaks4kbAround_fc_TN.sh

#!/bin/bash

#SBATCH --job-name=Peaks4kbAround_fc_TN
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=Peaks4kbAround_fc_TN.out
#SBATCH --error=Peaks4kbAround_fc_TN.err
#SBATCH --partition=broadwl
#SBATCH --mem=12G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

featureCounts -O -a /project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED_4kbaroundEnd.UNIQ.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/Peaks4kbAroundGeneEnd/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Peaks4kbAround.Total.fc /project2/gilad/briana/threeprimeseq/data/sort/*-T-*-sort.bam -s 2

featureCounts -O -a //project2/gilad/briana/threeprimeseq/data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed_RENAMED_4kbaroundEnd.UNIQ.SAF -F SAF -o /project2/gilad/briana/threeprimeseq/data/Peaks4kbAroundGeneEnd/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Peaks4kbAround.Nuclear.fc /project2/gilad/briana/threeprimeseq/data/sort/*-N-*-sort.bam -s 2

fix_head_fc_Peaks4kbAround_tot.py

infile= open("/project2/gilad/briana/threeprimeseq/data/Peaks4kbAroundGeneEnd/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Peaks4kbAround.Total.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/Peaks4kbAroundGeneEnd/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Peaks4kbAround.Total_fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

fix_head_fc_gPeaks4kbAround_nuc.py

infile= open("/project2/gilad/briana/threeprimeseq/data/Peaks4kbAroundGeneEnd/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Peaks4kbAround.Nuclear.fc", "r")
fout = open("/project2/gilad/briana/threeprimeseq/data/Peaks4kbAroundGeneEnd/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Peaks4kbAround.Nuclear_fixed.fc",'w')
for line, i in enumerate(infile):
    if line == 1:
        i_list=i.split()
        libraries=i_list[:6]
        for sample in i_list[6:]:
            full = sample.split("/")[7]
            samp= full.split("-")[2:4]
            lim="_"
            samp_st=lim.join(samp)
            libraries.append(samp_st)
        first_line= "\t".join(libraries)
        fout.write(first_line + '\n')
    else :
        fout.write(i)
fout.close()

Use no filter and standardization scheme:

total_Cov_18486_4kb=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Peaks4kbAround.Total_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_T)%>%  group_by(gene) %>% filter(X18486_T>10) %>% summarize(GeneSum=sum(X18486_T)) %>% mutate(GeneSumNorm=GeneSum/10.8) %>% inner_join(geneLengths, by="gene") %>% mutate(GeneSumSt=GeneSum/length)

Join the data frames.

TXN_NormGene_4kb=TXN_abund %>% inner_join(total_Cov_18486_4kb,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene_4kb_n=TXN_NormGene_4kb %>% filter(TPM>0) %>% filter(GeneSumSt>0)
corr_18486Tot_noOver4kb=ggplot(TXN_NormGene_4kb_n, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Total", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.34") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red')

#+geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Tot_noOver4kb

Expand here to see past versions of unnamed-chunk-65-1.png:
Version Author Date
c3fd2c4 Briana Mittleman 2018-12-13 

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene_4kb_n)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene_4kb_n)

Residuals:
     Min       1Q   Median       3Q      Max 
-3.07128 -0.26438  0.05285  0.31624  2.23108 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)      2.200525   0.013284  165.65   <2e-16 ***
log10(GeneSumSt) 0.409498   0.005733   71.43   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.5116 on 9738 degrees of freedom
Multiple R-squared:  0.3438,    Adjusted R-squared:  0.3437 
F-statistic:  5102 on 1 and 9738 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_4kb_n$TPM),log10(TXN_NormGene_4kb_n$GeneSumSt),method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_4kb_n$TPM),
log10(TXN_NormGene_4kb_n$GeneSumSt), : Cannot compute exact p-value with
ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_4kb_n$TPM) and log10(TXN_NormGene_4kb_n$GeneSumSt)
S = 6.5566e+10, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.5742507 
nuclear_Cov_18486_4kb=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_Peaks4kbAround.Nuclear_fixed.fc", header=T, stringsAsFactors = F)[,1:7] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18486_N) %>%  group_by(gene) %>%filter(X18486_N>10) %>%  summarize(GeneSum=sum(X18486_N)) %>% mutate(GeneSumNorm=GeneSum/11.4) %>% inner_join(geneLengths, by="gene") %>% mutate(GeneSumSt=GeneSum/length)

Join the data frames.

TXN_NormGene_noOverlap_4kb=TXN_abund %>% inner_join(nuclear_Cov_18486_4kb,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene_noOverlap_4kb_n=TXN_NormGene_noOverlap_4kb %>% filter(TPM>0) %>% filter(GeneSumSt>0)
corr_18486Nuc_4kb=ggplot(TXN_NormGene_noOverlap_4kb_n, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Nuclear", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.26") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red')

#+ geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18486Nuc_4kb

Expand here to see past versions of unnamed-chunk-68-1.png:
Version Author Date
c3fd2c4 Briana Mittleman 2018-12-13 

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene_noOverlap_4kb_n)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene_noOverlap_4kb_n)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.3104 -0.3004  0.0465  0.3451  2.2798 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)      2.203466   0.016119  136.70   <2e-16 ***
log10(GeneSumSt) 0.402256   0.006733   59.74   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.5572 on 10000 degrees of freedom
Multiple R-squared:  0.263, Adjusted R-squared:  0.2629 
F-statistic:  3569 on 1 and 10000 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlap_4kb_n$TPM),log10(TXN_NormGene_noOverlap_4kb_n$GeneSumSt),method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_noOverlap_4kb_n$TPM),
log10(TXN_NormGene_noOverlap_4kb_n$GeneSumSt), : Cannot compute exact p-
value with ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_noOverlap_4kb_n$TPM) and log10(TXN_NormGene_noOverlap_4kb_n$GeneSumSt)
S = 8.3048e+10, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
     rho 
0.502012

From here I can subset down to just the genes in the nonoverlapping set.

TXN_NormGene_noOverlap_4kb_noOver=TXN_NormGene_noOverlap_4kb_n %>% semi_join(TXN_NormGene_noOverlapEXT, by="gene")


corr_18486Nuc_4kb_noOVer=ggplot(TXN_NormGene_noOverlap_4kb_noOver, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Nuclear", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.27")


#+geom_text(aes(label=gene),hjust=0, vjust=0)+geom_density2d(na.rm = TRUE, size = 1, colour = 'red')
       
corr_18486Nuc_4kb_noOVer 

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene_noOverlap_4kb_noOver))

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene_noOverlap_4kb_noOver)

Residuals:
     Min       1Q   Median       3Q      Max 
-2.61075 -0.31776  0.04514  0.34370  2.19168 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)       2.27850    0.04047   56.30   <2e-16 ***
log10(GeneSumSt)  0.41540    0.01509   27.53   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.5504 on 2006 degrees of freedom
Multiple R-squared:  0.2742,    Adjusted R-squared:  0.2739 
F-statistic: 757.9 on 1 and 2006 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlap_4kb_noOver$TPM),log10(TXN_NormGene_noOverlap_4kb_noOver$GeneSumSt) ,method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_noOverlap_4kb_noOver$TPM), :
Cannot compute exact p-value with ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_noOverlap_4kb_noOver$TPM) and log10(TXN_NormGene_noOverlap_4kb_noOver$GeneSumSt)
S = 664430000, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.5076096

Look at curves for the TPM and standards.

 ggplot(TXN_NormGene_noOverlap_4kb_noOver, aes(x=log10(GeneSumSt))) + geom_density()

ggplot(TXN_NormGene_noOverlap_4kb_noOver, aes(x=log10(TPM))) + geom_density()

Separate by percentile gene expression

Check another line

total_Cov_18497_noOver=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlap.Total_fixed.fc", header=T, stringsAsFactors = F)[,1:8] %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") %>% select(gene, X18497_T)%>%  group_by(gene) %>% summarize(GeneSum=sum(X18497_T)) %>% mutate(GeneSumNorm=GeneSum/10.8) %>% inner_join(geneLengths, by="gene") %>% mutate(GeneSumSt=GeneSum/length)

Join the data frames.

TXN_NormGene_noOverlap_497=TXN_abund %>% inner_join(total_Cov_18497_noOver,by="gene")

Remove rows with 0 counts and Plot:

TXN_NormGene_noOverlap_497=TXN_NormGene_noOverlap_497 %>% filter(TPM>0) %>% filter(GeneSumSt>0)
corr_18497Tot_noOver=ggplot(TXN_NormGene_noOverlap_497, aes(x=log10(TPM), y= log10(GeneSumSt))) + geom_point() + labs(title="Total", x="log10 RNA seq TPM", y="log10 Peak count sum per gene")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumSt)),method = "lm") + annotate("text",x=3, y=5,label="R2=.49") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') 

#+ geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_18497Tot_noOver       

summary(lm(log10(TPM)~log10(GeneSumSt),TXN_NormGene_noOverlap_497)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumSt), data = TXN_NormGene_noOverlap_497)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.5597 -0.2929  0.0903  0.3993  2.8953 

Coefficients:
                 Estimate Std. Error t value Pr(>|t|)    
(Intercept)      2.413566   0.015014   160.8   <2e-16 ***
log10(GeneSumSt) 0.650761   0.006389   101.9   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.6346 on 10739 degrees of freedom
Multiple R-squared:  0.4914,    Adjusted R-squared:  0.4913 
F-statistic: 1.037e+04 on 1 and 10739 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_NormGene_noOverlap_497$TPM),log10(TXN_NormGene_noOverlap_497$GeneSumSt),method="spearman")
Warning in cor.test.default(log10(TXN_NormGene_noOverlap_497$TPM),
log10(TXN_NormGene_noOverlap_497$GeneSumSt), : Cannot compute exact p-value
with ties

    Spearman's rank correlation rho

data:  log10(TXN_NormGene_noOverlap_497$TPM) and log10(TXN_NormGene_noOverlap_497$GeneSumSt)
S = 6.3783e+10, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.6911702

This shows me the effect is not line specific because the correlation is the same when i use the RNA from one line and 3’ from another.

Sum over all individals

First I will get the total counts for the non overlapping set:

TotCounts_nonOverlapEX=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total_fixed.fc", header=T, stringsAsFactors = F)[,7:45]


SumCounts_Tot=rowSums(TotCounts_nonOverlapEX)

Alllib_Tot_nonOverlapEX=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total_fixed.fc", header=T, stringsAsFactors = F) %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") 
 
Alllib_Tot_nonOverlapEX$SumCounts=SumCounts_Tot

Alllib_Tot_nonOverlapEX_bygene=Alllib_Tot_nonOverlapEX %>% select(gene, SumCounts) %>%  group_by(gene)  %>%  summarize(GeneSum=sum(SumCounts)) %>% mutate(GeneSumNorm=GeneSum/11.4) 


TXN_abund_combLibs_tot=TXN_abund %>% inner_join(Alllib_Tot_nonOverlapEX_bygene,by="gene")


TXN_abund_combLibs_tot_n0=TXN_abund_combLibs_tot %>% filter(TPM>0) %>% filter(GeneSumNorm>0)
corr_AllLibTot_noOver=ggplot(TXN_abund_combLibs_tot_n0, aes(x=log10(TPM), y= log10(GeneSumNorm))) + geom_point() + labs(title="Total All ind", x="log10 RNA seq TPM", y="log10 Peak count sum per gene All Ind.")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumNorm)),method = "lm") + annotate("text",x=-2, y=5,label="R2=.55") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') + geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_AllLibTot_noOver       

summary(lm(log10(TPM)~log10(GeneSumNorm),TXN_abund_combLibs_tot_n0)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumNorm), data = TXN_abund_combLibs_tot_n0)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.2653 -0.3200  0.0183  0.3435  3.4494 

Coefficients:
                   Estimate Std. Error t value Pr(>|t|)    
(Intercept)        -1.34124    0.03578  -37.48   <2e-16 ***
log10(GeneSumNorm)  0.78488    0.01279   61.38   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.6682 on 3079 degrees of freedom
Multiple R-squared:  0.5503,    Adjusted R-squared:  0.5501 
F-statistic:  3767 on 1 and 3079 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_abund_combLibs_tot_n0$TPM),log10(TXN_abund_combLibs_tot_n0$GeneSumNorm),method="spearman")
Warning in cor.test.default(log10(TXN_abund_combLibs_tot_n0$TPM),
log10(TXN_abund_combLibs_tot_n0$GeneSumNorm), : Cannot compute exact p-
value with ties

    Spearman's rank correlation rho

data:  log10(TXN_abund_combLibs_tot_n0$TPM) and log10(TXN_abund_combLibs_tot_n0$GeneSumNorm)
S = 1.211e+09, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.7515536

Same for nuclear

NucCounts_nonOverlapEX=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Nuclear_Fixed.fc", header=T, stringsAsFactors = F)[,7:45]


SumCounts_Nuc=rowSums(NucCounts_nonOverlapEX)

Alllib_Nuc_nonOverlapEX=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Nuclear_Fixed.fc", header=T, stringsAsFactors = F) %>% separate(Geneid, into=c("peak", "chr", "start", "end", "strand", "gene"), sep=":") 
 
Alllib_Nuc_nonOverlapEX$SumCounts=SumCounts_Nuc

Alllib_Nuc_nonOverlapEX_bygene=Alllib_Nuc_nonOverlapEX %>% select(gene, SumCounts) %>%  group_by(gene)  %>%  summarize(GeneSum=sum(SumCounts)) %>% mutate(GeneSumNorm=GeneSum/10.8) 


TXN_abund_combLibs_Nuc=TXN_abund %>% inner_join(Alllib_Nuc_nonOverlapEX_bygene,by="gene")


TXN_abund_combLibs_Nuc_n0=TXN_abund_combLibs_Nuc%>% filter(TPM>0) %>% filter(GeneSumNorm>0)
corr_AllLibNuc_noOver=ggplot(TXN_abund_combLibs_Nuc_n0, aes(x=log10(TPM), y= log10(GeneSumNorm))) + geom_point() + labs(title="Nuclear All ind", x="log10 RNA seq TPM", y="log10 Peak count sum per gene All Ind.")+ geom_smooth(aes(x=log10(TPM),y=log10(GeneSumNorm)),method = "lm") + annotate("text",x=-2, y=5,label="R2=.48") +geom_density2d(na.rm = TRUE, size = 1, colour = 'red') 

#+ geom_text(aes(label=gene),hjust=0, vjust=0)
       
corr_AllLibNuc_noOver       

summary(lm(log10(TPM)~log10(GeneSumNorm),TXN_abund_combLibs_Nuc_n0)) 

Call:
lm(formula = log10(TPM) ~ log10(GeneSumNorm), data = TXN_abund_combLibs_Nuc_n0)

Residuals:
    Min      1Q  Median      3Q     Max 
-3.3980 -0.3912  0.0130  0.4109  3.3698 

Coefficients:
                   Estimate Std. Error t value Pr(>|t|)    
(Intercept)        -1.40221    0.04238  -33.08   <2e-16 ***
log10(GeneSumNorm)  0.75348    0.01427   52.78   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.7219 on 3079 degrees of freedom
Multiple R-squared:  0.475, Adjusted R-squared:  0.4749 
F-statistic:  2786 on 1 and 3079 DF,  p-value: < 2.2e-16
cor.test(log10(TXN_abund_combLibs_Nuc_n0$TPM),log10(TXN_abund_combLibs_Nuc_n0$GeneSumNorm),method="spearman")
Warning in cor.test.default(log10(TXN_abund_combLibs_Nuc_n0$TPM),
log10(TXN_abund_combLibs_Nuc_n0$GeneSumNorm), : Cannot compute exact p-
value with ties

    Spearman's rank correlation rho

data:  log10(TXN_abund_combLibs_Nuc_n0$TPM) and log10(TXN_abund_combLibs_Nuc_n0$GeneSumNorm)
S = 1587500000, p-value < 2.2e-16
alternative hypothesis: true rho is not equal to 0
sample estimates:
      rho 
0.6743196

Compare 2 3’ Seq.

TotCounts_nonOverlapEX=read.table("../data/UnderstandPeaksQC/filtered_APApeaks_merged_allchrom_refseqGenes.Transcript_sm_quant_GenicNoOverlapEXT.Total_fixed.fc", header=T, stringsAsFactors = F)

summary(lm(TotCounts_nonOverlapEX$X18486_T~TotCounts_nonOverlapEX$X19238_T))

Call:
lm(formula = TotCounts_nonOverlapEX$X18486_T ~ TotCounts_nonOverlapEX$X19238_T)

Residuals:
     Min       1Q   Median       3Q      Max 
-10543.0     -2.6      1.3      1.3  11904.2 

Coefficients:
                                  Estimate Std. Error t value Pr(>|t|)    
(Intercept)                     -1.2603937  0.3532729  -3.568  0.00036 ***
TotCounts_nonOverlapEX$X19238_T  0.7807420  0.0009045 863.135  < 2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 101.2 on 82478 degrees of freedom
Multiple R-squared:  0.9003,    Adjusted R-squared:  0.9003 
F-statistic: 7.45e+05 on 1 and 82478 DF,  p-value: < 2.2e-16
summary(lm(TotCounts_nonOverlapEX$X18486_T~TotCounts_nonOverlapEX$X19238_T))

Call:
lm(formula = TotCounts_nonOverlapEX$X18486_T ~ TotCounts_nonOverlapEX$X19238_T)

Residuals:
     Min       1Q   Median       3Q      Max 
-10543.0     -2.6      1.3      1.3  11904.2 

Coefficients:
                                  Estimate Std. Error t value Pr(>|t|)    
(Intercept)                     -1.2603937  0.3532729  -3.568  0.00036 ***
TotCounts_nonOverlapEX$X19238_T  0.7807420  0.0009045 863.135  < 2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 101.2 on 82478 degrees of freedom
Multiple R-squared:  0.9003,    Adjusted R-squared:  0.9003 
F-statistic: 7.45e+05 on 1 and 82478 DF,  p-value: < 2.2e-16
summary(lm(TotCounts_nonOverlapEX$X19238_T~TotCounts_nonOverlapEX$X19225_T))

Call:
lm(formula = TotCounts_nonOverlapEX$X19238_T ~ TotCounts_nonOverlapEX$X19225_T)

Residuals:
     Min       1Q   Median       3Q      Max 
-13986.1     -7.7     -6.1     -2.1  12207.9 

Coefficients:
                                Estimate Std. Error t value Pr(>|t|)    
(Intercept)                     6.146796   0.454280   13.53   <2e-16 ***
TotCounts_nonOverlapEX$X19225_T 0.837322   0.001034  809.62   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 130.3 on 82478 degrees of freedom
Multiple R-squared:  0.8882,    Adjusted R-squared:  0.8882 
F-statistic: 6.555e+05 on 1 and 82478 DF,  p-value: < 2.2e-16
plot(log10(TotCounts_nonOverlapEX$X19238_T)~log10(TotCounts_nonOverlapEX$X19225_T))

Session information

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  tximport_1.8.0  cowplot_0.9.3   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] modelr_0.1.2      readxl_1.1.0      bindr_0.1.1      
[16] plyr_1.8.4        munsell_0.5.0     gtable_0.2.0     
[19] cellranger_1.1.0  rvest_0.3.2       R.methodsS3_1.7.1
[22] evaluate_0.11     labeling_0.3      knitr_1.20       
[25] broom_0.5.0       Rcpp_0.12.19      scales_1.0.0     
[28] backports_1.1.2   jsonlite_1.5      hms_0.4.2        
[31] digest_0.6.17     stringi_1.2.4     grid_3.5.1       
[34] rprojroot_1.3-2   cli_1.0.1         tools_3.5.1      
[37] magrittr_1.5      lazyeval_0.2.1    crayon_1.3.4     
[40] whisker_0.3-2     pkgconfig_2.0.2   MASS_7.3-50      
[43] xml2_1.2.0        lubridate_1.7.4   assertthat_0.2.0 
[46] rmarkdown_1.10    httr_1.3.1        rstudioapi_0.8   
[49] R6_2.3.0          nlme_3.1-137      git2r_0.23.0     
[52] compiler_3.5.1

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