Last updated: 2018-10-24

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    File Version Author Date Message
    Rmd 96a97f4 Briana Mittleman 2018-10-24 add nuclear characterization


This analysis is similar to the Characterize Total APAqtl analysis

I would like to study:

  • Distance metrics:
    • distance from snp to TSS of gene
    • Distance from snp to peak
  • Expression metrics:
    • expression of genes with significant QTLs vs other genes (by rna seq)
    • expression of genes with significant QTLs vs other genes (peak coverage)
  • Chrom HMM metrics:
    • look at the chrom HMM interval for the significant QTLs

Upload Libraries and Data:

Library

library(workflowr)
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library(reshape2)
library(tidyverse)
── Attaching packages ─────────────────────────────────────────────────────────────── tidyverse 1.2.1 ──
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✔ tidyr   0.8.1     ✔ stringr 1.3.1
✔ readr   1.1.1     ✔ forcats 0.3.0
── Conflicts ────────────────────────────────────────────────────────────────── tidyverse_conflicts() ──
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library(VennDiagram)
Loading required package: grid
Loading required package: futile.logger
library(data.table)

Attaching package: 'data.table'
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library(cowplot)

Attaching package: 'cowplot'
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Permuted Results from APA:

I will add a column to this dataframe that will tell me if the association is significant at 10% FDR. This will help me plot based on significance later in the analysis. I am also going to seperate the PID into relevant pieces.

NuclearAPA=read.table("../data/perm_QTL_trans/filtered_APApeaks_merged_allchrom_refseqGenes_pheno_Nuclear_transcript_permResBH.txt", stringsAsFactors = F, header=T)  %>% mutate(sig=ifelse(-log10(bh)>=1, 1,0 )) %>%  separate(pid, sep = ":", into=c("chr", "start", "end", "id")) %>% separate(id, sep = "_", into=c("gene", "strand", "peak"))

NuclearAPA$sig=as.factor(NuclearAPA$sig)


print(names(NuclearAPA))
 [1] "chr"    "start"  "end"    "gene"   "strand" "peak"   "nvar"  
 [8] "shape1" "shape2" "dummy"  "sid"    "dist"   "npval"  "slope" 
[15] "ppval"  "bpval"  "bh"     "sig"   

Distance Metrics

Distance from snp to TSS

I ran the QTL analysis based on the starting position of the gene.

ggplot(NuclearAPA, aes(x=dist, fill=sig, by=sig)) + geom_density(alpha=.5)  +  labs(title="Distance from snp to TSS", x="Base Pairs") + scale_fill_discrete(guide = guide_legend(title = "Significant QTL")) + scale_fill_brewer(palette="Paired")
Scale for 'fill' is already present. Adding another scale for 'fill',
which will replace the existing scale.

Zoom in to 100,000.

ggplot(NuclearAPA, aes(x=dist, fill=sig, by=sig)) + geom_density(alpha=.5)+coord_cartesian(xlim = c(-150000, 150000)) + scale_fill_brewer(palette="Paired")

### Distance from snp to peak

To perform this analysis I need to recover the peak positions.

The peak file I used for the QTL analysis is: /project2/gilad/briana/threeprimeseq/data/mergedPeaks_comb/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed.bed

peaks=read.table("../data/PeaksUsed/filtered_APApeaks_merged_allchrom_refseqTrans.noties_sm.fixed.bed", col.names = c("chr", "peakStart", "peakEnd", "PeakNum", "PeakScore", "Strand", "Gene")) %>% mutate(peak=paste("peak", PeakNum,sep="")) %>% mutate(PeakCenter=peakStart+ (peakEnd- peakStart))

I want to join the peak start to the NuclearAPA file but the peak column. I will then create a column that is snppos-peakcenter.

NuclearAPA_peakdist= NuclearAPA %>%  inner_join(peaks, by="peak") %>%  separate(sid, into=c("snpCHR", "snpLOC"), by=":")
NuclearAPA_peakdist$snpLOC= as.numeric(NuclearAPA_peakdist$snpLOC)

NuclearAPA_peakdist= NuclearAPA_peakdist %>%  mutate(DisttoPeak= snpLOC-PeakCenter)

Plot this by significance.

ggplot(NuclearAPA_peakdist, aes(x=DisttoPeak, fill=sig, by=sig)) + geom_density(alpha=.5)  +  labs(title="Distance from snp peak", x="log10 absolute value Distance to Peak") + scale_fill_discrete(guide = guide_legend(title = "Significant QTL")) + scale_fill_brewer(palette="Paired")
Scale for 'fill' is already present. Adding another scale for 'fill',
which will replace the existing scale.

Look at the summarys based on significance:

NuclearAPA_peakdist_sig=NuclearAPA_peakdist %>% filter(sig==1)
NuclearAPA_peakdist_notsig=NuclearAPA_peakdist %>% filter(sig==0)


summary(NuclearAPA_peakdist_sig$DisttoPeak)
    Min.  1st Qu.   Median     Mean  3rd Qu.     Max. 
-1003786   -17579      -91    -8818     6588   891734 
summary(NuclearAPA_peakdist_notsig$DisttoPeak)
     Min.   1st Qu.    Median      Mean   3rd Qu.      Max. 
-70147526   -265059     -2067      7263    255169 125172864 
ggplot(NuclearAPA_peakdist, aes(y=DisttoPeak,x=sig, fill=sig, by=sig)) + geom_boxplot()  + scale_fill_discrete(guide = guide_legend(title = "Significant QTL")) + scale_fill_brewer(palette="Paired")
Scale for 'fill' is already present. Adding another scale for 'fill',
which will replace the existing scale.

Look like there are some outliers that are really far. I will remove variants greater than 1*10^6th away

NuclearAPA_peakdist_filt=NuclearAPA_peakdist %>% filter(abs(DisttoPeak) <= 1*(10^6))

ggplot(NuclearAPA_peakdist_filt, aes(y=DisttoPeak,x=sig, fill=sig, by=sig)) + geom_boxplot()  + scale_fill_discrete(guide = guide_legend(title = "Significant QTL")) + facet_grid(.~strand) + scale_fill_brewer(palette="Paired")
Scale for 'fill' is already present. Adding another scale for 'fill',
which will replace the existing scale.

ggplot(NuclearAPA_peakdist_filt, aes(x=DisttoPeak, fill=sig, by=sig)) + geom_density()  + scale_fill_discrete(guide = guide_legend(title = "Significant QTL")) + facet_grid(.~strand)+ scale_fill_brewer(palette="Paired")
Scale for 'fill' is already present. Adding another scale for 'fill',
which will replace the existing scale.

I am going to plot a violin plot for just the significant ones.

ggplot(NuclearAPA_peakdist_sig, aes(x=DisttoPeak)) + geom_density()

Within 1000 bases of the peak center.

NuclearAPA_peakdist_sig %>% filter(abs(DisttoPeak) < 1000) %>% nrow()
[1] 192
NuclearAPA_peakdist_sig %>% filter(abs(DisttoPeak) < 10000) %>% nrow()
[1] 420
NuclearAPA_peakdist_sig %>% filter(abs(DisttoPeak) < 100000) %>% nrow()
[1] 726

192 QTLs are within 1000 bp, 420 are within 10000, and 726 are within 100,000bp

Expression metrics

Next I want to pull in the expression values and compare the expression of genes with a nuclear APA qtl in comparison to genes without one. I will also need to pull in the gene names file to add in the gene names from the ensg ID.

Remove the # from the file.

expression=read.table("../data/mol_pheno/fastqtl_qqnorm_RNAseq_phase2.fixed.noChr.txt", header = T,stringsAsFactors = F)
expression_mean=apply(expression[,5:73],1,mean,na.rm=TRUE)
expression_var=apply(expression[,5:73],1,var,na.rm=TRUE)
expression$exp.mean= expression_mean 
expression$exp.var=expression_var
expression= expression %>% separate(ID, into=c("Gene.stable.ID", "ver"), sep ="[.]")

Now I can pull in the names and join the dataframes.

geneNames=read.table("../data/ensemble_to_genename.txt", sep="\t", header=T,stringsAsFactors = F) 

expression=expression %>% inner_join(geneNames,by="Gene.stable.ID") 

expression=expression %>% select(Chr, start, end, Gene.name, exp.mean,exp.var) %>%  rename("gene"=Gene.name)

Now I can join this with the qtls.

NuclearAPA_wExp=NuclearAPA %>% inner_join(expression, by="gene") 
gene_wQTL_N= NuclearAPA_wExp %>% group_by(gene) %>% summarise(sig_gene=sum(as.numeric(as.character(sig)))) %>% ungroup() %>% inner_join(expression, by="gene") %>% mutate(sigGeneFactor=ifelse(sig_gene>=1, 1,0))

gene_wQTL_N$sigGeneFactor= as.factor(as.character(gene_wQTL_N$sigGeneFactor))
summary(gene_wQTL_N$sigGeneFactor)
   0    1 
4589  607 

There are 607 genes with a QTL

ggplot(gene_wQTL_N, aes(x=exp.mean, by=sigGeneFactor, fill=sigGeneFactor)) + geom_density(alpha=.3) +labs(title="Mean in RNA expression by genes with significant QTL", x="Mean in normalized expression") + scale_fill_discrete(guide = guide_legend(title = "Significant QTL"))+ scale_fill_brewer(palette="Paired")
Scale for 'fill' is already present. Adding another scale for 'fill',
which will replace the existing scale.

I can do a similar analysis but test the variance in the gene expression.

ggplot(gene_wQTL_N, aes(x=exp.var, by=sigGeneFactor, fill=sigGeneFactor)) + geom_density(alpha=.3) + labs(title="Varriance in RNA expression by genes with significant QTL", x="Variance in normalized expression") + scale_fill_discrete(guide = guide_legend(title = "Significant QTL"))+ scale_fill_brewer(palette="Paired")
Scale for 'fill' is already present. Adding another scale for 'fill',
which will replace the existing scale.

Peak coverage for QTLs

I can also look at peak coverage for peaks with QLTs and those without. I will first look at this on peak level then mvoe to gene level. The peak scores come from the coverage in the peaks.

The NuclearAPA_peakdist data frame has the information I need for this.

ggplot(NuclearAPA_peakdist, aes(x=PeakScore,fill=sig,by=sig)) + geom_density(alpha=.5)+ scale_x_log10() + labs(title="Peak score by significance") + scale_fill_brewer(palette="Paired")

This is expected. It makes sense that we have more power to detect qtls in higher expressed peaks. This leads me to believe that filtering out low peaks may add power but will not mitigate the effect. ##Where are the SNPs

I created the significant SNP files in the Characterize Total APAqtl analysis analysis.

chromHmm=read.table("../data/ChromHmmOverlap/chromHMM_regions.txt", col.names = c("number", "name"), stringsAsFactors = F)

NuclearOverlapHMM=read.table("../data/ChromHmmOverlap/Nuc_overlapHMM.bed", col.names=c("chrom", "start", "end", "sid", "significance", "strand", "number"))
NuclearOverlapHMM$number=as.integer(NuclearOverlapHMM$number)
NuclearOverlapHMM_names=NuclearOverlapHMM %>% left_join(chromHmm, by="number")
ggplot(NuclearOverlapHMM_names, aes(x=name)) + geom_bar() + labs(title="ChromHMM labels for Nuclear APAQtls" , y="Number of SNPs", x="Region")+theme(axis.text.x = element_text(angle = 90, hjust = 1))

I do still need to get 880 random snps.

shuf -n 880 /project2/gilad/briana/threeprimeseq/data/nominal_APAqtl_trans/filtered_APApeaks_merged_allchrom_refseqGenes_pheno_Nuclear_NomRes.txt > /project2/gilad/briana/threeprimeseq/data/nominal_APAqtl_trans/randomSnps/ApaQTL_nuclear_Random880.txt

Run QTLNOMres2SigSNPbed.py with nuclear 880 and sort output

import pybedtools 

RANDnuc=pybedtools.BedTool('/project2/gilad/briana/threeprimeseq/data/nominal_APAqtl_trans/randomSnps/ApaQTL_nuclear_Random880.sort.bed') 



hmm=pybedtools.BedTool("/project2/gilad/briana/genome_anotation_data/GM12878.chromHMM.sort.bed")

#map hmm to snps  
NucRnad_overlapHMM=RANDnuc.map(hmm, c=4)


#save results  

NucRnad_overlapHMM.saveas("/project2/gilad/briana/threeprimeseq/data/nominal_APAqtl_trans/randomSnps/ApaQTL_nuclear_Random_overlapHMM.bed")
NuclearRandOverlapHMM=read.table("../data/ChromHmmOverlap/ApaQTL_nuclear_Random_overlapHMM.bed", col.names=c("chrom", "start", "end", "sid", "significance", "strand", "number"))

NuclearRandOverlapHMM_names=NuclearRandOverlapHMM %>% left_join(chromHmm, by="number")
ggplot(NuclearRandOverlapHMM_names, aes(x=name)) + geom_bar() + labs(title="ChromHMM labels for Nuclear APAQtls (Random)" , y="Number of SNPs", x="Region")+theme(axis.text.x = element_text(angle = 90, hjust = 1))

To put this on the same plot I can count the number in each then plot them next to eachother.

random_perChromHMM_nuc=NuclearRandOverlapHMM_names %>%  group_by(name) %>% summarise(Random=n())
sig_perChromHMM_nuc= NuclearOverlapHMM_names %>%  group_by(name) %>%  summarise(Nuclear_QTLs=n())

perChrommHMM_nuc=random_perChromHMM_nuc %>%  full_join(sig_perChromHMM_nuc, by="name", ) %>% replace_na(list(Random=0,Total_QTLs=0))  

perChrommHMM_nuc_melt=melt(perChrommHMM_nuc, id.vars="name")
names(perChrommHMM_nuc_melt)=c("Region","Set", "N_Snps" )
chromenrichNuclearplot=ggplot(perChrommHMM_nuc_melt, aes(x=Region, y=N_Snps, by=Set, fill=Set)) + geom_bar(position="dodge", stat="identity") +theme(axis.text.x = element_text(angle = 90, hjust = 1)) + labs(title="Enrichment of Nuclear QTLs by chromatin region", y="Number of Snps", x="Chromatin Region") + scale_fill_brewer(palette="Paired")
chromenrichNuclearplot

ggsave("../output/plots/ChromHmmEnrich_Nuclear.png", chromenrichNuclearplot)
Saving 7 x 5 in image

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] grid      stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
 [1] bindrcpp_0.2.2      cowplot_0.9.3       data.table_1.11.8  
 [4] VennDiagram_1.6.20  futile.logger_1.4.3 forcats_0.3.0      
 [7] stringr_1.3.1       dplyr_0.7.6         purrr_0.2.5        
[10] readr_1.1.1         tidyr_0.8.1         tibble_1.4.2       
[13] ggplot2_3.0.0       tidyverse_1.2.1     reshape2_1.4.3     
[16] workflowr_1.1.1    

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   lambda.r_1.2.3       modelr_0.1.2        
[16] readxl_1.1.0         bindr_0.1.1          plyr_1.8.4          
[19] munsell_0.5.0        gtable_0.2.0         cellranger_1.1.0    
[22] rvest_0.3.2          R.methodsS3_1.7.1    evaluate_0.11       
[25] labeling_0.3         knitr_1.20           broom_0.5.0         
[28] Rcpp_0.12.19         formatR_1.5          backports_1.1.2     
[31] scales_1.0.0         jsonlite_1.5         hms_0.4.2           
[34] digest_0.6.17        stringi_1.2.4        rprojroot_1.3-2     
[37] cli_1.0.1            tools_3.5.1          magrittr_1.5        
[40] lazyeval_0.2.1       futile.options_1.0.1 crayon_1.3.4        
[43] whisker_0.3-2        pkgconfig_2.0.2      xml2_1.2.0          
[46] lubridate_1.7.4      assertthat_0.2.0     rmarkdown_1.10      
[49] httr_1.3.1           rstudioapi_0.8       R6_2.3.0            
[52] nlme_3.1-137         git2r_0.23.0         compiler_3.5.1      



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