Yap KD profile
Reactome
TFBS TSS
TFBS ENH
suppressPackageStartupMessages({
library("tidyverse")
library("reshape2")
library("DESeq2")
library("gplots")
library("fgsea")
library("MASS")
library("mitch")
})
Here is the Yap knock-down data wee generated previously.
x <- read.table("paired-edgeR.xls",header=TRUE,row.names=1)
head(x)
## logFC logCPM LR PValue
## ENSMUSG00000031880_Rrad 3.213046 3.8147728 97.23076 6.170060e-23
## ENSMUSG00000097418_Mir155hg 2.073579 5.4823122 67.78697 1.821492e-16
## ENSMUSG00000068614_Actc1 -1.191931 8.1779897 59.15924 1.454148e-14
## ENSMUSG00000023043_Krt18 2.668002 2.1851742 59.08167 1.512616e-14
## ENSMUSG00000053110_Yap1 -1.128658 5.6493272 58.21689 2.347550e-14
## ENSMUSG00000057400_Ces1c -6.104755 0.7602132 38.15317 6.540352e-10
## adj.p.value Dispersion totreads predLFC1
## ENSMUSG00000031880_Rrad 8.141394e-19 0.05215232 1041 3.177282
## ENSMUSG00000097418_Mir155hg 1.201729e-12 0.03796428 3341 2.068754
## ENSMUSG00000068614_Actc1 4.989743e-11 0.01638376 21540 -1.191579
## ENSMUSG00000023043_Krt18 4.989743e-11 0.03579011 324 2.572206
## ENSMUSG00000053110_Yap1 6.195184e-11 0.01355845 3716 -1.126844
## ENSMUSG00000057400_Ces1c 1.438332e-06 0.11883120 113 -4.319255
## predLFC3 predLFC5 Y_YAP1 Y_YAP2 Y_YAP3
## ENSMUSG00000031880_Rrad 3.099560 3.027000 4.414797 4.131310 5.188433
## ENSMUSG00000097418_Mir155hg 2.057827 2.047042 6.214675 6.702181 5.303606
## ENSMUSG00000068614_Actc1 -1.190775 -1.189973 6.965110 7.992973 7.331905
## ENSMUSG00000023043_Krt18 2.379678 2.218070 3.514757 -1.997335 3.493719
## ENSMUSG00000053110_Yap1 -1.122720 -1.118627 4.999306 4.578748 5.303606
## ENSMUSG00000057400_Ces1c -2.846752 -2.233624 -5.633764 -3.367146 -5.633764
## C_C1 C_C2 C_C3 Y_YAP1.1 Y_YAP2.1
## ENSMUSG00000031880_Rrad 2.1810119 0.9584748 0.8019469 254 228
## ENSMUSG00000097418_Mir155hg 3.5479142 4.2223398 4.2523728 885 1356
## ENSMUSG00000068614_Actc1 8.5485078 8.9090382 8.4132372 1489 3318
## ENSMUSG00000023043_Krt18 0.9170423 -3.2687604 0.6593102 136 3
## ENSMUSG00000053110_Yap1 6.1546449 6.0295060 6.0954641 381 311
## ENSMUSG00000057400_Ces1c -5.6337641 -0.5373202 3.0967067 0 1
## Y_YAP3.1 C_C1.1 C_C2.1 C_C3.1
## ENSMUSG00000031880_Rrad 457 58 23 21
## ENSMUSG00000097418_Mir155hg 495 150 223 232
## ENSMUSG00000068614_Actc1 2020 4810 5749 4154
## ENSMUSG00000023043_Krt18 141 24 1 19
## ENSMUSG00000053110_Yap1 495 915 781 833
## ENSMUSG00000057400_Ces1c 0 0 8 104
sig <- subset(x,adj.p.value<0.05)
plot(x$logCPM, x$logFC, col="gray", xlab="logCPM",ylab="log2 fold change")
points(sig$logCPM, sig$logFC, col="red")
We will perform analysis using Reactome gene sets as well as Cistrome transcription factor targets (promoter and enhancer).
download.file("https://reactome.org/download/current/ReactomePathways.gmt.zip", destfile="ReactomePathways.gmt.zip")
unzip("ReactomePathways.gmt.zip")
reactome <- gmt_import("ReactomePathways.gmt")
tft_tss <- gmt_import("MouseTfPeaks.gmt")
tft_enh <- gmt_import("MouseTfPeaks_enh.gmt")
Lets look at the make up of the gene set library. Reactome has more smaller sets while TFT targets has mostly 1000 genes.
length(reactome)
## [1] 2400
length(tft_tss)
## [1] 9054
length(tft_enh)
## [1] 9055
summary(unname(unlist(lapply(reactome,length))))
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 1.00 5.00 15.00 49.03 43.00 2825.00
summary(unname(unlist(lapply(tft_tss,length))))
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 1.0 187.0 1000.0 691.8 1000.0 1000.0
summary(unname(unlist(lapply(tft_enh,length))))
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 1 1000 1000 916 1000 1000
Here we will run the mitch package with reactome gene sets (human). Reactome gives a broad brush analysis of major biochemical and signaling pathways.
rownames(x) <- sapply(strsplit(rownames(x),"_"),"[[",1)
m2h <- read.table("mouse2human.txt.sort")
m2h[,1]=NULL
# import
m <- mitch_import(x=x,DEtype="edger",geneTable=m2h)
## The input is a single dataframe; one contrast only. Converting
## it to a list for you.
## Note: Mean no. genes in input = 13195
## Note: no. genes in output = 11694
## Note: estimated proportion of input genes in output = 0.886
# calc
capture.output(
res <- mitch_calc(x=m,genesets=reactome,priority="effect")
, file = "/dev/null", append = FALSE,
type = c("output", "message"), split = FALSE)
## Note: Enrichments with large effect sizes may not be
## statistically significant.
head(res$enrichment_result,20)
## set setSize
## 488 Initial triggering of complement 12
## 993 Signal regulatory protein family interactions 13
## 1236 Xenobiotics 14
## 163 Chemokine receptors bind chemokines 16
## 823 RIP-mediated NFkB activation via ZBP1 17
## 757 Plasma lipoprotein assembly 15
## 519 Interleukin-4 and Interleukin-13 signaling 73
## 202 Cristae formation 10
## 150 Cell recruitment (pro-inflammatory response) 17
## 803 Purinergic signaling in leishmaniasis infection 17
## 41 Activation of Matrix Metalloproteinases 15
## 44 Activation of PPARGC1A (PGC-1alpha) by phosphorylation 10
## 1140 The NLRP3 inflammasome 13
## 510 Interleukin-10 signaling 19
## 267 Diseases associated with the TLR signaling cascade 19
## 268 Diseases of Immune System 19
## 357 Formation of Senescence-Associated Heterochromatin Foci (SAHF) 10
## 1238 ZBP1(DAI) mediated induction of type I IFNs 20
## 237 Defective B4GALT7 causes EDS, progeroid type 15
## 518 Interleukin-37 signaling 17
## pANOVA s.dist p.adjustANOVA
## 488 1.087822e-05 0.7333077 9.239236e-04
## 993 1.702453e-05 0.6888504 1.355578e-03
## 1236 1.941340e-05 -0.6593200 1.454863e-03
## 163 2.477303e-04 0.5292056 9.912677e-03
## 823 1.627449e-04 0.5282934 7.149551e-03
## 757 7.080126e-04 -0.5050775 2.031764e-02
## 519 1.791109e-13 0.4986167 1.140937e-10
## 202 7.291862e-03 -0.4900719 9.676908e-02
## 150 5.077529e-04 0.4871064 1.617193e-02
## 803 5.077529e-04 0.4871064 1.617193e-02
## 41 2.188838e-03 0.4569056 4.316931e-02
## 44 1.367372e-02 -0.4503595 1.417995e-01
## 1140 6.061536e-03 0.4396818 8.303652e-02
## 510 9.137808e-04 0.4394906 2.476929e-02
## 267 1.136361e-03 0.4313535 2.680970e-02
## 268 1.136361e-03 0.4313535 2.680970e-02
## 357 1.846133e-02 0.4303663 1.603145e-01
## 1238 1.003077e-03 0.4250043 2.583913e-02
## 237 4.798947e-03 0.4206582 7.113707e-02
## 518 2.915456e-03 0.4170642 5.088070e-02
# report
unlink("yaprna_reactome.html")
capture.output(
mitch_report(res, "yaprna_reactome.html")
, file = "/dev/null", append = FALSE,
type = c("output", "message"), split = FALSE)
## Dataset saved as " /tmp/RtmpukgUOB/./yaprna_reactome.RData ".
##
##
## processing file: mitch.Rmd
## Contour plot does not apply to unidimensional analysis.
## output file: /mnt/bfx6/bfx/kevinwatt/yap_kd/mitch/mitch.knit.md
##
## Output created: /tmp/RtmpukgUOB/mitch_report.html
Here I'm going to run TFBS enrichment analysis based on TSS binding.
# set up gene table
x <- read.table("paired-edgeR.xls",header=TRUE,row.names=1)
gt <- data.frame(rownames(x))
gt$gname <- sapply(strsplit(gt[,1],"_"),"[[",2)
# import
m <- mitch_import(x=x,DEtype="edger",geneTable=gt)
## The input is a single dataframe; one contrast only. Converting
## it to a list for you.
## Note: Mean no. genes in input = 13195
## Note: no. genes in output = 13193
## Note: estimated proportion of input genes in output = 1
# calc
capture.output(
res <- mitch_calc(x=m,genesets=tft_tss,priority="effect",minsetsize=50)
, file = "/dev/null", append = FALSE,
type = c("output", "message"), split = FALSE)
## Note: Enrichments with large effect sizes may not be
## statistically significant.
head(res$enrichment_result,20)
## set setSize pANOVA s.dist
## 6786 STAT4_None_NaturalKillerCell_86782 80 2.866500e-05 0.2708228
## 3655 BCL6_None_Hepatocyte_5856 143 6.646365e-08 -0.2620422
## 4099 RNF2_None_EmbryonicStemCell_68313 51 1.741390e-03 0.2536039
## 838 PER2_None_Epithelium_37826 53 1.977680e-03 -0.2458086
## 920 STAT1_None_Macrophage_38373 68 8.846188e-04 0.2333625
## 763 RELA_None_Macrophage_37417 619 1.675613e-20 0.2202755
## 6339 EP300_None_Macrophage_83658 529 5.817833e-17 0.2141554
## 3570 NEUROD1_R1_EmbryonicStemCell_58027 89 8.828038e-04 0.2041799
## 2522 FOSB_None_CorticalNeuron_49586 57 8.226172e-03 0.2025183
## 6543 STAT6_None_Macrophage_85093 575 2.056471e-16 0.2021030
## 6452 JUNB_MIN_Macrophage_84498 486 3.547982e-14 0.2019823
## 1379 AR_None_None_41602 96 6.419736e-04 0.2018369
## 6052 POU5F1_None_EmbryonicFibroblast_81810 58 8.387325e-03 0.2002730
## 6211 IRF4_None_Treg_82830 50 1.516701e-02 0.1986487
## 6996 FOS_MIN_Macrophage_88308 615 9.635147e-17 0.1978791
## 6865 EP300_None_Macrophage_87483 602 5.980800e-16 0.1946338
## 4528 RELA_None_Macrophage_70691 617 3.038181e-16 0.1943152
## 783 RELA_None_Macrophage_37420 623 3.304342e-16 0.1931841
## 6337 EP300_None_Macrophage_83657 540 3.190790e-14 0.1923736
## 2710 NLRC5_None_TLymphocyte_51237 60 1.010146e-02 0.1921622
## p.adjustANOVA
## 6786 5.502256e-04
## 3655 3.332478e-06
## 4099 1.321245e-02
## 838 1.444483e-02
## 920 7.735021e-03
## 763 1.201414e-16
## 6339 1.390462e-13
## 3570 7.728576e-03
## 2522 4.369011e-02
## 6543 2.948979e-13
## 6452 1.211382e-11
## 1379 6.080516e-03
## 6052 4.422218e-02
## 6211 6.817470e-02
## 6996 1.727100e-13
## 6865 5.360292e-13
## 4528 3.384591e-13
## 783 3.384591e-13
## 6337 1.211060e-11
## 2710 5.089774e-02
unlink("yaprna_tss.html")
capture.output(
mitch_report(res, "yaprna_tss.html")
, file = "/dev/null", append = FALSE,
type = c("output", "message"), split = FALSE)
## Dataset saved as " /tmp/RtmpukgUOB/./yaprna_tss.RData ".
##
##
## processing file: mitch.Rmd
## Contour plot does not apply to unidimensional analysis.
## output file: /mnt/bfx6/bfx/kevinwatt/yap_kd/mitch/mitch.knit.md
##
## Output created: /tmp/RtmpukgUOB/mitch_report.html
Here I'm going to run TFBS enrichment analysis based on enhancer binding.
# calc
capture.output(
res <- mitch_calc(x=m,genesets=tft_enh,priority="effect",minsetsize=50)
, file = "/dev/null", append = FALSE,
type = c("output", "message"), split = FALSE)
## Note: Enrichments with large effect sizes may not be
## statistically significant.
head(res$enrichment_result,20)
## set setSize pANOVA
## 8405 MECP2_None_None_87515 53 1.432209e-02
## 8352 CXXC1_Mouseembryonicstemcells_EmbryonicStemCell_87252 52 1.928949e-02
## 8035 SPEN_MIN_None_85440 50 3.307592e-02
## 7522 CXXC1_Mouseembryonicstemcells_EmbryonicStemCell_82995 58 5.420931e-02
## 5304 RELA_None_Th1_69834 552 2.131003e-08
## 8349 CXXC1_Mouseembryonicstemcells_EmbryonicStemCell_87249 50 8.985524e-02
## 8327 POLR2A_Cone_M_Y-257.03_iPSC_87099 82 3.688276e-02
## 8404 MECP2_None_None_87514 72 5.399709e-02
## 8441 STAT3_None_Hepatocyte_87718 91 3.480773e-02
## 241 BMI1_None_Astrocyte_32783 80 6.215504e-02
## 312 SRSF2_None_EmbryonicFibroblast_33050 152 1.377207e-02
## 8403 EP300_None_Macrophage_87481 547 4.744125e-06
## 3953 CREB1_None_Macrophage_55168 81 7.732811e-02
## 8350 CXXC1_Mouseembryonicstemcells_EmbryonicStemCell_87251 73 9.646052e-02
## 8285 MED12_None_EmbryonicStemCell_86791 527 1.172950e-05
## 4916 RNF2_None_EmbryonicStemCell_68315 94 6.033290e-02
## 5487 SPI1_None_Macrophage_70701 578 5.198124e-06
## 1793 RUNX3_None_TLymphocyte_41959 69 1.085194e-01
## 8449 STAT3_None_Hepatocyte_87722 55 1.556160e-01
## 2413 RAG2_None_Thymocyte_4505 540 1.416120e-05
## s.dist p.adjustANOVA
## 8405 -0.1946124 0.1687192223
## 8352 -0.1877096 0.1903545261
## 8035 -0.1743407 0.2383253953
## 7522 -0.1462715 0.2876132447
## 5304 0.1405402 0.0001875709
## 8349 -0.1387598 0.3553426900
## 8327 0.1334831 0.2492971036
## 8404 -0.1314686 0.2870062923
## 8441 0.1281828 0.2431783599
## 241 0.1207676 0.3029283788
## 312 0.1160157 0.1649564083
## 8403 0.1153391 0.0129170112
## 3953 0.1136684 0.3337972107
## 8350 -0.1126420 0.3670335024
## 8285 0.1124596 0.0129170112
## 4916 0.1122580 0.2986059804
## 5487 0.1118710 0.0129170112
## 1793 -0.1118451 0.3870992035
## 8449 0.1107820 0.4505696104
## 2413 0.1101103 0.0129170112
unlink("yaprna_enh.html")
capture.output(
mitch_report(res, "yaprna_enh.html")
, file = "/dev/null", append = FALSE,
type = c("output", "message"), split = FALSE)
## Dataset saved as " /tmp/RtmpukgUOB/./yaprna_enh.RData ".
##
##
## processing file: mitch.Rmd
## Contour plot does not apply to unidimensional analysis.
## output file: /mnt/bfx6/bfx/kevinwatt/yap_kd/mitch/mitch.knit.md
##
## Output created: /tmp/RtmpukgUOB/mitch_report.html