Last updated: 2019-05-06

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File	Version	Author	Date	Message
Rmd	1d8a0a3	brimittleman	2019-05-06	add res
html	60093ce	brimittleman	2019-05-02	Build site.
Rmd	24c2ceb	brimittleman	2019-05-02	add diff iso

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()

In this analysis I wil use leafcutter to call PAS with differential ussage between fractions.

Prepare annotation

I first filter the annotated peak SAF file for peaks passing the 5% coverage in either fraction.

python makeSAFbothfrac5perc.py

Peak quantification

mkdir bothFrac_FC

Run feature counts with these peaks with both fractions:

sbatch bothFrac_FC.sh

Fix the header:

python fixFChead_bothfrac.py ../data/bothFrac_FC/APApeaks.ALLChrom.Filtered.Named.GeneLocAnnoPARSED.5percCov.bothfrac.fc ../data/bothFrac_FC/APApeaks.ALLChrom.Filtered.Named.GeneLocAnnoPARSED.5percCov.bothfrac.fixed.fc

Remove location demoniaiton:

Prepare leafcutter phenotype

mkdir ../data/DiffIso

python fc2leafphen.py

Fix pheno to remove location:

python removeloc_pheno.py ../data/DiffIso/APApeaks.ALLChrom.Filtered.Named.GeneLocAnnoPARSED.5percCov.bothfrac.fixed.forLC.fc ../data/DiffIso/APApeaks.ALLChrom.Filtered.Named.GeneLocAnnoPARSED.5percCov.bothfrac.fixed.forLC_noloc.fc

python subset_diffisopheno.py 1
python subset_diffisopheno.py 2
python subset_diffisopheno.py 3
python subset_diffisopheno.py 4
python subset_diffisopheno.py 5
python subset_diffisopheno.py 6
python subset_diffisopheno.py 7
python subset_diffisopheno.py 8
python subset_diffisopheno.py 9
python subset_diffisopheno.py 10
python subset_diffisopheno.py 11
python subset_diffisopheno.py 12
python subset_diffisopheno.py 13
python subset_diffisopheno.py 14
python subset_diffisopheno.py 15
python subset_diffisopheno.py 16
python subset_diffisopheno.py 18
python subset_diffisopheno.py 19
python subset_diffisopheno.py 20
python subset_diffisopheno.py 21
python subset_diffisopheno.py 22

Make the sample groups file:

python LC_samplegroups.py

Run leafcutter

The leafcutter environment is not in the three-prime-seq environment. Make sure leafcutter is installed and working.

sbatch run_leafcutterDiffIso.sh

Concatinate results:

awk '{if(NR>1)print}' ../data/DiffIso/TN_diff_isoform_chr*.txt_effect_sizes.txt > ../data/DiffIso/TN_diff_isoform_allChrom.txt_effect_sizes.txt


awk '{if(NR>1)print}' ../data/DiffIso/TN_diff_isoform_chr*.txt_cluster_significance.txt > ../data/DiffIso/TN_diff_isoform_AllChrom_cluster_significance.txt

Evaluate results

Significant clusters

sig=read.table("../data/DiffIso/TN_diff_isoform_AllChrom_cluster_significance.txt",sep="\t" ,col.names = c('status','loglr','df','p','cluster','p.adjust'),stringsAsFactors = F) %>% filter(status=="Success")

sig$p.adjust=as.numeric(as.character(sig$p.adjust))

qqplot(-log10(runif(nrow(sig))), -log10(sig$p.adjust),ylab="-log10 Total Adjusted Leafcutter pvalue", xlab="-log 10 Uniform expectation", main="Leafcutter differencial isoform analysis between fractions")
abline(0,1)

tested_genes=nrow(sig)
tested_genes

[1] 10815

sig_genes=sig %>% filter(p.adjust<.05)
number_sig_genes=nrow(sig_genes)
number_sig_genes

[1] 9446

Effect sizes

effectsize=read.table("../data/DiffIso/TN_diff_isoform_AllChrom_effect_sizes.txt", stringsAsFactors = F, col.names=c('intron',  'logef' ,'Nuclear', 'Total','deltaPAU')) %>% filter(intron != "intron")

effectsize$deltaPAU=as.numeric(as.character(effectsize$deltaPAU))
effectsize$logef=as.numeric(as.character(effectsize$logef))

Plot delta PAU:

plot(sort(effectsize$deltaPAU),main="Leafcutter delta PAU", ylab="Delta PAU", xlab="PAS Index")

Filter PAU > .2

effectsize_deltaPAU= effectsize %>% filter(abs(deltaPAU) > .2) 
nrow(effectsize_deltaPAU)

[1] 2090

Genes in this set:

effectsize_deltaPAU_Genes= effectsize_deltaPAU %>% separate(intron, into=c("chrom", "start", "end","gene"),sep=":") %>% group_by(gene) %>% summarise(nperGene=n()) 

nrow(effectsize_deltaPAU_Genes)

[1] 1689

Filter >.2 in Nuclear

effectsize_deltaPAU_nuclear= effectsize_deltaPAU %>% filter(deltaPAU < 0)

FIlter >.2 in Total:

effectsize_deltaPAU_total= effectsize_deltaPAU %>% filter(deltaPAU > 0)

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_0.23.0     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

Total v Nuclear

Briana Mittleman

5/2/2019