Last updated: 2019-06-19

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Knit directory: apaQTL/analysis/

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Rmd 3e5cc8e brimittleman 2019-06-07 code up to FC

I am interested in finding examples of intronic PAS that show RNAseq signatures upstream of the PAS but not downstream. To do this I will create a ratio of reads upstream/reads downstream standardized by the length of the region (up/downstream).

To do this I can use the work I did previously. Here I assigned each intronic PAS to an intron. I will do this analysis with the total fraction because I will be looking at steady state RNA seq.

library(workflowr)
This is workflowr version 1.3.0
Run ?workflowr for help getting started
library(tidyverse)
── Attaching packages ────────────────────── tidyverse 1.2.1 ──
✔ ggplot2 3.1.1       ✔ purrr   0.3.2  
✔ tibble  2.1.1       ✔ dplyr   0.8.0.1
✔ tidyr   0.8.3       ✔ stringr 1.3.1  
✔ readr   1.3.1       ✔ forcats 0.3.0  
── Conflicts ───────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()

Total

mkdir ../data/intronRNAratio
totIntronicPeaks=read.table("../data/peaks_5perc/APApeak_Peaks_GeneLocAnno.Total.5perc.fc", stringsAsFactors = F, header = F,col.names = c("chr", "start", "end", "gene", "loc", "strand", "peak", "avgUsage")) %>% filter(loc=="intron") 

pas2intronTot=read.table("../data/intron_analysis/TotalIntronPeaksontoIntrons.bed",col.names = c("intronCHR", "intronStart", "intronEnd", "gene", "score", "strand", "peakCHR", "peakStart", "peakEnd", "PeakID", "meanUsage", "peakStrand"))  %>% mutate(PASloc=ifelse(strand=="+", peakEnd, peakStart)) %>% dplyr::select(intronCHR,intronStart, intronEnd, gene, strand, PeakID, PASloc ,meanUsage)

write.table(pas2intronTot, "../data/intronRNAratio/TotalIntronicPAS2Intron.txt", quote = F, row.names = F, col.names = F, sep="\t")

Make upstream and downstream PAS saf files using python.

 python getIntronUpstreamPAS.py
 python getIntronDownstreamPAS.py

These make Bed and SAF files. I will use the SAF files for feature counts with all of the RNA seq. These files are in /project2/yangili1/LCL/RNAseqGeuvadisBams/*.final.bam

sbatch FC_intornUpandDownsteamPAS.sh

Downstream Results:

downstream=read.table("../data/intronRNAratio/DownstreamIntron.fc", header = T,stringsAsFactors = F)
downstreamMean=rowSums(downstream[,7:ncol(downstream)])

downstreanMeanDF=as.data.frame(cbind(downstream[,1:6], downstreamMean)) %>% mutate(DownstreamMean_st=downstreamMean/Length) %>% select(Geneid,DownstreamMean_st )

Upstream Results:

upstream=read.table("../data/intronRNAratio/UpstreamIntron.fc", header = T,stringsAsFactors = F)
upstreamMean=rowSums(upstream[,7:ncol(upstream)])
upstreamMeanDF=as.data.frame(cbind(upstream[,1:6], upstreamMean)) %>% mutate(UpstreamMean_st=upstreamMean/Length) %>% select(Geneid,UpstreamMean_st )

Join Results:

I will use upstream - downstream

pas2intronTot_peaks=pas2intronTot %>% separate(PeakID, into=c("PAS", "gene", "loc"), sep=":") %>% select(PAS)


UpandDown=upstreamMeanDF %>% inner_join(downstreanMeanDF, by="Geneid") %>% mutate(UpMinusDown=UpstreamMean_st-DownstreamMean_st) %>% arrange(desc(UpMinusDown)) %>% separate(Geneid, sep=":", into=c("PAS", "gene", "loc", "PASloc", "Usage"))  %>% semi_join(pas2intronTot_peaks, by="PAS")


summary(UpandDown$UpMinusDown)
      Min.    1st Qu.     Median       Mean    3rd Qu.       Max. 
-1246.7926    -0.0324     0.0000     0.3251     0.2171   541.0908

I want to know how many are positive:

MoreUp=UpandDown %>% filter(UpMinusDown>0) 
summary(MoreUp$UpMinusDown)
    Min.  1st Qu.   Median     Mean  3rd Qu.     Max. 
  0.0000   0.0352   0.2268   2.7767   1.1271 541.0908 
nrow(MoreUp)
[1] 4131

4131 examples where there are more reads upstream in the intron than downstream.

To look for examples:

head(MoreUp)
        PAS     gene    loc    PASloc              Usage UpstreamMean_st
1  peak5519     HFM1 intron  91852934  0.889259259259259        541.0908
2 peak31203   NAP1L1 intron  76443346  0.906666666666667        473.3895
3 peak87311  BHLHE40 intron   5023296  0.866111111111111        321.8041
4 peak50349   ATXN2L intron  28844314 0.0661111111111111        217.0654
5  peak7377 NOTCH2NL intron 145277488  0.715925925925926        186.1226
6 peak60248   MYL12A intron   3248635  0.862777777777778        175.4362
  DownstreamMean_st UpMinusDown
1          0.000000    541.0908
2         99.452381    373.9371
3          6.033469    315.7707
4         14.939394    202.1260
5         12.959547    173.1631
6          9.109116    166.3271
write.table(MoreUp, file="../data/intronRNAratio/TotalPAS_MoreUpstreamRNAreads.txt", col.names = T, row.names = F, quote = F, sep="\t" )

Nuclear

We also have nacent RNA seq. I will do this with nuclear intronic PAS.

nucIntronicPeaks=read.table("../data/peaks_5perc/APApeak_Peaks_GeneLocAnno.Nuclear.5perc.fc", stringsAsFactors = F, header = F,col.names = c("chr", "start", "end", "gene", "loc", "strand", "peak", "avgUsage")) %>% filter(loc=="intron")


pas2intronNuc=read.table("../data/intron_analysis/IntronPeaksontoIntrons.bed",col.names = c("intronCHR", "intronStart", "intronEnd", "gene", "score", "strand", "peakCHR", "peakStart", "peakEnd", "PeakID", "meanUsage", "peakStrand"))  %>% mutate(PASloc=ifelse(strand=="+", peakEnd, peakStart)) %>% dplyr::select(intronCHR,intronStart, intronEnd, gene, strand, PeakID, PASloc ,meanUsage)

write.table(pas2intronNuc, "../data/intronRNAratio/NuclearIntronicPAS2Intron.txt", quote = F, row.names = F, col.names = F, sep="\t")

Make upstream and downstream PAS saf files using python.

python getUpstreamIntronNuclear.py
python getDownstreamIntronNuclear.py

These make Bed and SAF files. I will use the SAF files for feature counts with all of the nacent RNA seq.

sbatch FC_NucintornUpandDown.sh

Downstream Results:

downstreamNuc=read.table("../data/intronRNAratio/NuclearDownstreamIntron.fc", col.names = c("Geneid","Chr","Start", "End", "Strand", "Length", "Downstream"), header = T,stringsAsFactors = F)%>% mutate(Downstream_st=Downstream/Length) %>% select(Geneid,Downstream_st )

upstreamNuc=read.table("../data/intronRNAratio/NuclearUpstreamIntron.fc", col.names = c("Geneid","Chr","Start", "End", "Strand", "Length", "Upstream"), header = T,stringsAsFactors = F)%>% mutate(Upstream_st=Upstream/Length) %>% select(Geneid,Upstream_st )


pas2intronNucPAS=pas2intronNuc %>% separate(PeakID, into=c("PAS", "gene", "loc"), sep=":") %>% select(PAS)


UpandDown_nuc=upstreamNuc %>% inner_join(downstreamNuc, by="Geneid") %>% mutate(UpMinusDown=Upstream_st-Downstream_st) %>% arrange(desc(UpMinusDown)) %>% separate(Geneid, sep=":", into=c("PAS", "gene", "loc", "PASloc", "Usage")) %>% semi_join(pas2intronNucPAS, by="PAS")




summary(UpandDown_nuc$UpMinusDown)
      Min.    1st Qu.     Median       Mean    3rd Qu.       Max. 
-269.33846   -0.06775    0.01552    0.08418    0.36174   57.52867 
MoreUpNuc=UpandDown_nuc %>% filter(UpMinusDown>0) 
summary(MoreUpNuc$UpMinusDown)
    Min.  1st Qu.   Median     Mean  3rd Qu.     Max. 
 0.00001  0.08524  0.29229  0.66527  0.74025 57.52867 
nrow(MoreUpNuc)
[1] 7625
head(MoreUpNuc)
         PAS     gene    loc    PASloc              Usage Upstream_st
1  peak18198    BTAF1 intron  93719017  0.468148148148148    68.64486
2  peak83249 MIR155HG intron  26939933 0.0524074074074074    44.98583
3  peak86087    DDX17 intron  38887949 0.0955555555555556    89.22321
4   peak9172   SLAMF1 intron 160597898  0.123518518518519    17.37310
5  peak94269    RSRC1 intron 157921145 0.0548148148148148    18.57143
6 peak111306     IRF4 intron    403950 0.0694444444444444    15.43238
  Downstream_st UpMinusDown
1     11.116190    57.52867
2      0.000000    44.98583
3     57.814883    31.40833
4      0.000000    17.37310
5      2.275192    16.29624
6      0.000000    15.43238
write.table(MoreUpNuc, file="../data/intronRNAratio/NuclearPAS_MoreUpstreamNascentreads.txt", col.names = T, row.names = F, quote = F, sep="/t" )

sessionInfo()
R version 3.5.1 (2018-07-02)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Scientific Linux 7.4 (Nitrogen)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] forcats_0.3.0   stringr_1.3.1   dplyr_0.8.0.1   purrr_0.3.2    
 [5] readr_1.3.1     tidyr_0.8.3     tibble_2.1.1    ggplot2_3.1.1  
 [9] tidyverse_1.2.1 workflowr_1.3.0

loaded via a namespace (and not attached):
 [1] Rcpp_1.0.0       cellranger_1.1.0 pillar_1.3.1     compiler_3.5.1  
 [5] git2r_0.25.2     plyr_1.8.4       tools_3.5.1      digest_0.6.18   
 [9] lubridate_1.7.4  jsonlite_1.6     evaluate_0.12    nlme_3.1-137    
[13] gtable_0.2.0     lattice_0.20-38  pkgconfig_2.0.2  rlang_0.3.1     
[17] cli_1.0.1        rstudioapi_0.10  yaml_2.2.0       haven_1.1.2     
[21] withr_2.1.2      xml2_1.2.0       httr_1.3.1       knitr_1.20      
[25] hms_0.4.2        generics_0.0.2   fs_1.2.6         rprojroot_1.3-2 
[29] grid_3.5.1       tidyselect_0.2.5 glue_1.3.0       R6_2.3.0        
[33] readxl_1.1.0     rmarkdown_1.10   modelr_0.1.2     magrittr_1.5    
[37] whisker_0.3-2    backports_1.1.2  scales_1.0.0     htmltools_0.3.6 
[41] rvest_0.3.2      assertthat_0.2.0 colorspace_1.3-2 stringi_1.2.4   
[45] lazyeval_0.2.1   munsell_0.5.0    broom_0.5.1      crayon_1.3.4