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Modified: data/orthofinder/Orthogroups/Orthogroups.txt
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We start by loading all the required R packages.
#(install first from CRAN or Bioconductor)
library("knitr")
library("rmdformats")
library("tidyverse")
library("data.table")
library("DT") # for making interactive search table
library("plotly") # for interactive plots
library("ggthemes") # for theme_calc
library("reshape2")
library("RColorBrewer")
library("ggpubr")
library("ggplot2")
library("ggrepel")
library("kableExtra")
library("kableExtra")
library("multiWGCNA")
# Path for all species
workDir <- "/Users/maevatecher/Library/Mobile Documents/com~apple~CloudDocs/Documents/GitHub/locust-comparative-genomics/data"NEED AUDELIA AND BERT TO GIVE DETAILS ON THE PROCESS
In the folder data/behavioral_data, the excel sheet
/Raw_data/All species_50specimens_update10Minutes contains
the measured from behavioral assay for 50 individuals per species per
density condition. As we do not have the exact behavioral data for each
specimen we sequenced, we estimated the phenotype by using the
median value of each group for the following
measures:
Moved distance (MovedDistance) in cm.
Stimulus zone time (StimulusZoneTime) in
seconds.
Non-stimulus zone time (NonStimulusZoneTime) in
seconds.
Time on stimulus wall (TimeStimulusWall) in
seconds.
We created an input file with data traits with a single line being the sample name (e.g., SAMER_G_Crd_SRR11815246) and the tissue used (Head = 0 and Thorax = 1) and rearing condition (Solitary = 0 and Crowded = 1).
behavior_file <- file.path(workDir, "/behavioral_data/Sample_phenotypes_WGCNA.csv")
behavior_df <- read.table(behavior_file, sep = ",", header = TRUE, quote = "", fill = TRUE, stringsAsFactors = FALSE)
datatable(behavior_df, options = list(
pageLength = 10, # Set initial page length
scrollX = TRUE, # Enable horizontal scrolling
autoWidth = TRUE, # Adjust column width automatically
searchHighlight = TRUE # Highlight search matches
),
rownames = FALSE,
escape = FALSE # Allows HTML formatting in table cells
)We start by loading all the files containing the read count data, selecting the needed columns, and merging all into one table.
setwd("C:/Users/Bert/Documents/Work/Papers/Transcriptomics-behavior/Revamped paper/WGCNA")
SPIC_CT1_count <- read.table("Transrate_SPIC_CT1_idxstats.tabular")
SPIC_CT2_count <- read.table("Transrate_SPIC_CT2_idxstats.tabular")
SPIC_CT3_count <- read.table("Transrate_SPIC_CT3_idxstats.tabular")
SPIC_CT4_count <- read.table("Transrate_SPIC_CT4_idxstats.tabular")
SPIC_CT5_count <- read.table("Transrate_SPIC_CT5_idxstats.tabular")
SPIC_IT1_count <- read.table("Transrate_SPIC_IT1_idxstats.tabular")
SPIC_IT2_count <- read.table("Transrate_SPIC_IT2_idxstats.tabular")
SPIC_IT3_count <- read.table("Transrate_SPIC_IT3_idxstats.tabular")
SPIC_IT4_count <- read.table("Transrate_SPIC_IT4_idxstats.tabular")
SPIC_IT5_count <- read.table("Transrate_SPIC_IT5_idxstats.tabular")
SAME_CT1_count <- read.table("Transrate_SAME_CT1_idxstats.tabular")
SAME_CT2_count <- read.table("Transrate_SAME_CT2_idxstats.tabular")
SAME_CT3_count <- read.table("Transrate_SAME_CT3_idxstats.tabular")
SAME_CT4_count <- read.table("Transrate_SAME_CT4_idxstats.tabular")
SAME_CT5_count <- read.table("Transrate_SAME_CT5_idxstats.tabular")
SAME_IT1_count <- read.table("Transrate_SAME_IT1_idxstats.tabular")
SAME_IT2_count <- read.table("Transrate_SAME_IT2_idxstats.tabular")
SAME_IT3_count <- read.table("Transrate_SAME_IT4_idxstats.tabular")
SAME_IT4_count <- read.table("Transrate_SAME_IT5_idxstats.tabular")
SAME_IT5_count <- read.table("Transrate_SAME_IT6_idxstats.tabular")
SCUB_CT1_count <- read.table("Transrate_SCUB_CT1_idxstats.tabular")
SCUB_CT2_count <- read.table("Transrate_SCUB_CT2_idxstats.tabular")
SCUB_CT3_count <- read.table("Transrate_SCUB_CT3_idxstats.tabular")
SCUB_CT4_count <- read.table("Transrate_SCUB_CT4_idxstats.tabular")
SCUB_IT1_count <- read.table("Transrate_SCUB_IT1_idxstats.tabular")
SCUB_IT2_count <- read.table("Transrate_SCUB_IT2_idxstats.tabular")
SCUB_IT3_count <- read.table("Transrate_SCUB_IT3_idxstats.tabular")
SCUB_IT5_count <- read.table("Transrate_SCUB_IT5_idxstats.tabular")
SNIT_CT1_count <- read.table("Transrate_SNIT_CT1_idxstats.tabular")
SNIT_CT3_count <- read.table("Transrate_SNIT_CT3_idxstats.tabular")
SNIT_CT5_count <- read.table("Transrate_SNIT_CT5_idxstats.tabular")
SNIT_IT1_count <- read.table("Transrate_SNIT_IT1_idxstats.tabular")
SNIT_IT2_count <- read.table("Transrate_SNIT_IT2_idxstats.tabular")
SNIT_IT3_count <- read.table("Transrate_SNIT_IT3_idxstats.tabular")
SNIT_IT4_count <- read.table("Transrate_SNIT_IT4_idxstats.tabular")
SNIT_IT5_count <- read.table("Transrate_SNIT_IT5_idxstats.tabular")
mylist = do.call("list", mget(grep("count", ls(), value=T)))
mylist2 <- mylist[sapply(mylist, function(x) dim(x)[1]) > 0]
mylist2 <- lapply(mylist, function(x) { x <- x[c(1,3)] })
Complete <- mylist2[[1]]
colnames(Complete)[2] <- print(names(mylist2)[[1]])
for(i in 2:length(mylist2)){
Complete <- merge(Complete,mylist2[[i]], by = 'V1')
colnames(Complete)[i+1] <- print(names(mylist2)[[i]])
}Now we remove any genes with an average readcount under 5
Complete_low <- Complete
row.names(Complete_low) <- Complete_low$V1
Complete_low <- Complete_low[,-c(1)]
Complete_low <- subset(transform(Complete_low, Mean=round(rowMeans(Complete_low),1)), Mean >=5)
Complete_low <- Complete_low[,-c(37)]Now we convert our read counts to the TPM format for use in WGCNA
mylist_TPM<-mylist
for(i in 1:length(mylist_TPM)){
TPM_1 <- mylist_TPM[[i]]
TPM_1$x <- TPM_1$V3/TPM_1$V2
TPM <- TPM_1[c(5)]
row.names(TPM) <- TPM_1[,1]
TPM <- t(t(TPM)*1e6/colSums(TPM))
TPM <- as.data.frame(as.table(TPM))
mylist_TPM[[i]] <- TPM[c(1,3)]
}
TPM <- mylist_TPM[[1]]
colnames(TPM)[2] <- print(names(mylist_TPM)[[1]])
for(i in 2:length(mylist_TPM)){
TPM <- merge(TPM,mylist_TPM[[i]], by = 'Var1')
colnames(TPM)[i+1] <- print(names(mylist_TPM)[[i]])
}
Complete_low$Var1 <- row.names(Complete_low)
Complete_TPM <- subset(TPM, Var1 %in% Complete_low$Var1)
row.names(Complete_TPM) <- Complete_TPM$Var1
Complete_TPM <- Complete_TPM[-c(1)]Finally, we select the top 60 % of the most varying genes in the whole dataset and convert the dataframe for use in WGCNA
# Select most varying genes over the whole dataset
DE_genes <- apply(Complete_TPM, 1, var)
DE_genes <- names(sort(DE_genes, decreasing=TRUE))[1:(nrow(Complete_TPM)*0.6)]
Complete_DE <- Complete_TPM[DE_genes,]
datExpr <- as.data.frame(t(Complete_DE));
datExpr[length(mylist),] <- as.numeric(datExpr[length(mylist),])We first set the correct soft-thresholding power
powers = c(c(1:10), seq(from = 12, to=20, by=2))
sft = pickSoftThreshold(datExpr, powerVector = powers, verbose = 5)
sizeGrWindow(9, 5)
par(mfrow = c(1,2));
cex1 = 0.9;
plot(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2],
xlab="Soft Threshold (power)",ylab="Scale Free Topology Model Fit,signed R^2",type="n",
main = paste("Scale independence"));
text(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2],
labels=powers,cex=cex1,col="red");
abline(h=0.80,col="red")
plot(sft$fitIndices[,1], sft$fitIndices[,5],
xlab="Soft Threshold (power)",ylab="Mean Connectivity", type="n",
main = paste("Mean connectivity"))
text(sft$fitIndices[,1], sft$fitIndices[,5], labels=powers, cex=cex1,col="red")Based on these graphs, we select the power.We can now run the rest of WGCNA.
Power <- 8
CutHeight <- 0.2
net = blockwiseModules(datExpr, power = Power, maxBlockSize = 36000,
TOMType = "unsigned", minModuleSize = 30,
reassignThreshold = 0, mergeCutHeight = CutHeight,
numericLabels = TRUE, pamRespectsDendro = FALSE,
verbose = 3)
mergedColors = labels2colors(net$colors)
#PlotName <- paste0("Modules_", Var, "_", CutHeight , "_",ProjectName, "_",Sys.Date(),".pdf")
#pdf(PlotName)
plotDendroAndColors(net$dendrograms[[1]], mergedColors[net$blockGenes[[1]]],
"Module colors",
dendroLabels = FALSE, hang = 0.03,
addGuide = TRUE, guideHang = 0.05)
#dev.off()
moduleLabels = net$colors
moduleColors = labels2colors(net$colors)
MEs = net$MEs;
geneTree = net$dendrograms[[1]];We now import the trait data and run some tests.
# Import trait data
traitData = read.csv("C:/Users/Bert/Documents/Work/Papers/Transcriptomics-behavior/Revamped paper/WGCNA/datTraits_50samples_20_xi_2020_thorax.csv");
femaleSamples = rownames(datExpr);
traitRows = match(femaleSamples, traitData$Grasshopper);
datTraits = traitData[traitRows, -1];
rownames(datTraits) = traitData[traitRows, 1];
# PLOT SAMPLE DENDOGRAM AND TRAIT HEAT MAP
# Re-cluster samples
sampleTree2 = hclust(dist(datExpr), method = "average")
# Convert traits to a color representation: white means low, red means high, grey means missing entry
traitColors = numbers2colors(datTraits, signed = FALSE);
# Plot the sample dendrogram and the colors underneath.
plotDendroAndColors(sampleTree2, traitColors,
groupLabels = names(datTraits),
main = "Sample dendrogram and trait heatmap")We now calculate the correlations between the phenotypic traits and the modules of co-expressed genes
nGenes = ncol(datExpr);
nSamples = nrow(datExpr);
MEs0 = moduleEigengenes(datExpr, moduleColors)$eigengenes
MEs = orderMEs(MEs0)
moduleTraitCor = cor(MEs, datTraits, use = "p");
moduleTraitPvalue = corPvalueStudent(moduleTraitCor, nSamples)
textMatrix = paste(signif(moduleTraitCor, 2), "\n(",
signif(moduleTraitPvalue, 1), ")", sep = "");
dim(textMatrix) = dim(moduleTraitCor)
par(mar = c(6, 8.5, 3, 3));
#PlotName <- paste0("Correlations3_", Var, "_", CutHeight , "_",ProjectName, "_",Sys.Date(),".pdf")
#pdf(PlotName)
labeledHeatmap(Matrix = moduleTraitCor,
xLabels = names(datTraits),
yLabels = names(MEs),
ySymbols = names(MEs),
colorLabels = TRUE,
colors = blueWhiteRed(50),
textMatrix = textMatrix,
setStdMargins = FALSE,
cex.text = 0.14,
zlim = c(-1,1),
xLabelsAngle = 90,
yLabelsAngle = 45,
font.lab.x = 0.5,
font.lab.y = 0.5,
cex.lab.x = 0.5,
cex.lab.y = 0.4,
main = paste("Module-trait relationships")
)
#dev.off()We can now show the relationship between the different modules.
MEs = moduleEigengenes(datExpr, moduleColors)$eigengenes
Distance = as.data.frame(datTraits$Moved.distance);
names(Distance) = "moved.distance"
MET = orderMEs(cbind(MEs, Distance))
sizeGrWindow(5,7.5);
par(cex = 0.9)
plotEigengeneNetworks(MET, "", marDendro = c(0,4,1,2), marHeatmap = c(3,4,1,2), cex.lab = 0.8, xLabelsAngle
= 90)
sizeGrWindow(6,6);
par(cex = 1.0)
plotEigengeneNetworks(MET, "Eigengene dendrogram", marDendro = c(0,4,2,0),
plotHeatmaps = FALSE)
par(cex = 1.0)
plotEigengeneNetworks(MET, "Eigengene adjacency heatmap", marHeatmap = c(3,4,2,2),
plotDendrograms = FALSE, xLabelsAngle = 90)Now we export the different modules.
Trait1 = as.data.frame(datTraits$Stimulus.zone.time);
names(Trait1) = "Stimulus zone time"
Trait2 = as.data.frame(datTraits$Non.stimulus.zone.time);
names(Trait2) = "Non stimulus zone time"
Trait3 = as.data.frame(datTraits$Stimulus.wall.time);
names(Trait3) = "Time on stimulus wall"
Trait4 = as.data.frame(datTraits$Moved.distance);
names(Trait4) = "Moved distance"
Trait5 = as.data.frame(datTraits$Female.pronotum.length);
names(Trait5) = "Pronotum length"
Trait6 = as.data.frame(datTraits$Female.F.C);
names(Trait6) = "F/C"
Trait7 = as.data.frame(datTraits$Black.patterning.PCA1);
names(Trait7) = "PC1 Black patterns"
geneTraitSignificance1 = as.data.frame(cor(datExpr, Trait1, use = "p"));
geneTraitSignificance2 = as.data.frame(cor(datExpr, Trait2, use = "p"));
geneTraitSignificance3 = as.data.frame(cor(datExpr, Trait3, use = "p"));
geneTraitSignificance4 = as.data.frame(cor(datExpr, Trait4, use = "p"));
geneTraitSignificance5 = as.data.frame(cor(datExpr, Trait5, use = "p"));
geneTraitSignificance6 = as.data.frame(cor(datExpr, Trait6, use = "p"));
geneTraitSignificance7 = as.data.frame(cor(datExpr, Trait7, use = "p"));
GSPvalue1 = as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance1), nSamples));
GSPvalue2 = as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance2), nSamples));
GSPvalue3 = as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance3), nSamples));
GSPvalue4 = as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance4), nSamples));
GSPvalue5 = as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance5), nSamples));
GSPvalue6 = as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance6), nSamples));
GSPvalue7 = as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance7), nSamples));
names(geneTraitSignificance1) = paste("GS.", names(Trait1), sep="");
names(GSPvalue1) = paste("p.GS.", names(Trait1), sep="");
names(geneTraitSignificance2) = paste("GS.", names(Trait2), sep="");
names(GSPvalue2) = paste("p.GS.", names(Trait2), sep="");
names(geneTraitSignificance3) = paste("GS.", names(Trait3), sep="");
names(GSPvalue3) = paste("p.GS.", names(Trait3), sep="");
names(geneTraitSignificance4) = paste("GS.", names(Trait4), sep="");
names(GSPvalue4) = paste("p.GS.", names(Trait4), sep="");
names(geneTraitSignificance5) = paste("GS.", names(Trait5), sep="");
names(GSPvalue5) = paste("p.GS.", names(Trait5), sep="");
names(geneTraitSignificance6) = paste("GS.", names(Trait6), sep="");
names(GSPvalue6) = paste("p.GS.", names(Trait6), sep="");
names(geneTraitSignificance7) = paste("GS.", names(Trait7), sep="");
names(GSPvalue7) = paste("p.GS.", names(Trait7), sep="");
modNames = substring(names(MEs), 3)
geneModuleMembership = as.data.frame(cor(datExpr, MEs, use = "p"));
MMPvalue = as.data.frame(corPvalueStudent(as.matrix(geneModuleMembership), nSamples));
names(geneModuleMembership) = paste("MM", modNames, sep="");
names(MMPvalue) = paste("p.MM", modNames, sep="");
geneInfo0 = data.frame(moduleColor = moduleColors,
geneTraitSignificance1, GSPvalue1,
geneTraitSignificance2, GSPvalue2,
geneTraitSignificance3, GSPvalue3,
geneTraitSignificance4, GSPvalue4,
geneTraitSignificance5, GSPvalue5,
geneTraitSignificance6, GSPvalue6,
geneTraitSignificance7, GSPvalue7)
modOrder = order(-abs(cor(MEs, Trait1, use = "p")));
for (mod in 1:ncol(geneModuleMembership)){
oldNames = names(geneInfo0)
geneInfo0 = data.frame(geneInfo0, geneModuleMembership[, modOrder[mod]],
MMPvalue[, modOrder[mod]]);
names(geneInfo0) = c(oldNames, paste("MM.", modNames[modOrder[mod]], sep=""),
paste("p.MM.", modNames[modOrder[mod]], sep=""))
}
geneOrder = order(geneInfo0$moduleColor, -abs(geneInfo0[2]));
geneInfo = geneInfo0[geneOrder, ]
#TableName <- paste0("geneInfo2_", CutHeight , "_", ".txt")
#write.table(geneInfo,TableName,row.names = TRUE, col.names = TRUE, quote = FALSE,sep = '\t')
#for(i in 1:length(modNames)){
# TableName <- paste0(modNames[i], "_", length(which(moduleColors==modNames[i])), "genes_", CutHeight , "_",ProjectName, ".txt")
# write.table(names(datExpr)[moduleColors==modNames[i]],TableName, row.names=FALSE, col.names=FALSE, quote = FALSE)
#}Finally, we export data for input into VisANT. We are only showing it for the module cyan.
TOM = TOMsimilarityFromExpr(datExpr, power = 6);
module = "cyan";
probes = names(datExpr)
inModule = (moduleColors==module);
modProbes = probes[inModule];
modTOM = TOM[inModule, inModule];
dimnames(modTOM) = list(modProbes, modProbes)
vis = exportNetworkToVisANT(modTOM,
file = paste("VisANTInput-", module, ".txt", sep=""),
weighted = TRUE,
threshold = 0)
sessionInfo()R version 4.4.1 (2024-06-14)
Platform: aarch64-apple-darwin20
Running under: macOS 15.1
Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.12.0
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
time zone: Asia/Tokyo
tzcode source: internal
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] multiWGCNA_1.2.0 ggalluvial_0.12.5 kableExtra_1.4.0 ggrepel_0.9.6
[5] ggpubr_0.6.0 RColorBrewer_1.1-3 reshape2_1.4.4 ggthemes_5.1.0
[9] plotly_4.10.4 DT_0.33 data.table_1.16.2 lubridate_1.9.3
[13] forcats_1.0.0 stringr_1.5.1 dplyr_1.1.4 purrr_1.0.2
[17] readr_2.1.5 tidyr_1.3.1 tibble_3.2.1 ggplot2_3.5.1
[21] tidyverse_2.0.0 rmdformats_1.0.4 knitr_1.49
loaded via a namespace (and not attached):
[1] rstudioapi_0.17.1 jsonlite_1.8.9
[3] magrittr_2.0.3 farver_2.1.2
[5] rmarkdown_2.29 fs_1.6.5
[7] zlibbioc_1.50.0 vctrs_0.6.5
[9] memoise_2.0.1 base64enc_0.1-3
[11] rstatix_0.7.2 htmltools_0.5.8.1
[13] S4Arrays_1.4.1 dynamicTreeCut_1.63-1
[15] broom_1.0.7 SparseArray_1.4.8
[17] Formula_1.2-5 sass_0.4.9
[19] bslib_0.8.0 htmlwidgets_1.6.4
[21] plyr_1.8.9 impute_1.78.0
[23] cachem_1.1.0 igraph_2.1.1
[25] whisker_0.4.1 lifecycle_1.0.4
[27] iterators_1.0.14 pkgconfig_2.0.3
[29] Matrix_1.7-1 R6_2.5.1
[31] fastmap_1.2.0 GenomeInfoDbData_1.2.12
[33] MatrixGenerics_1.16.0 digest_0.6.37
[35] colorspace_2.1-1 patchwork_1.3.0
[37] AnnotationDbi_1.66.0 S4Vectors_0.42.1
[39] rprojroot_2.0.4 crosstalk_1.2.1
[41] Hmisc_5.2-1 GenomicRanges_1.56.2
[43] RSQLite_2.3.9 fansi_1.0.6
[45] timechange_0.3.0 httr_1.4.7
[47] abind_1.4-8 compiler_4.4.1
[49] rngtools_1.5.2 bit64_4.5.2
[51] withr_3.0.2 doParallel_1.0.17
[53] htmlTable_2.4.3 backports_1.5.0
[55] carData_3.0-5 DBI_1.2.3
[57] ggsignif_0.6.4 DelayedArray_0.30.1
[59] flashClust_1.01-2 tools_4.4.1
[61] foreign_0.8-87 httpuv_1.6.15
[63] nnet_7.3-19 glue_1.8.0
[65] promises_1.3.2 grid_4.4.1
[67] checkmate_2.3.2 cluster_2.1.6
[69] generics_0.1.3 gtable_0.3.6
[71] tzdb_0.4.0 preprocessCore_1.66.0
[73] hms_1.1.3 WGCNA_1.73
[75] xml2_1.3.6 car_3.1-3
[77] utf8_1.2.4 XVector_0.44.0
[79] BiocGenerics_0.50.0 foreach_1.5.2
[81] pillar_1.9.0 later_1.4.1
[83] splines_4.4.1 lattice_0.22-6
[85] survival_3.7-0 bit_4.5.0.1
[87] tidyselect_1.2.1 GO.db_3.19.1
[89] Biostrings_2.72.1 git2r_0.35.0
[91] gridExtra_2.3 bookdown_0.41
[93] IRanges_2.38.1 SummarizedExperiment_1.34.0
[95] svglite_2.1.3 stats4_4.4.1
[97] xfun_0.49 Biobase_2.64.0
[99] matrixStats_1.4.1 stringi_1.8.4
[101] UCSC.utils_1.0.0 workflowr_1.7.1
[103] lazyeval_0.2.2 yaml_2.3.10
[105] evaluate_1.0.1 codetools_0.2-20
[107] cli_3.6.3 rpart_4.1.23
[109] systemfonts_1.1.0 munsell_0.5.1
[111] jquerylib_0.1.4 Rcpp_1.0.13-1
[113] GenomeInfoDb_1.40.1 png_0.1-8
[115] fastcluster_1.2.6 parallel_4.4.1
[117] blob_1.2.4 dcanr_1.20.0
[119] doRNG_1.8.6 viridisLite_0.4.2
[121] scales_1.3.0 crayon_1.5.3
[123] rlang_1.1.4 cowplot_1.1.3
[125] KEGGREST_1.44.1