# HG changeset patch # User galaxyp # Date 1536942133 14400 # Node ID f1db758949f49b71f49bac0c1aa4f993b34c66e8 planemo upload commit a0e968c7bd2b6f7b963baeecb08f3a39e50f52d6 diff -r 000000000000 -r f1db758949f4 quantp.r --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/quantp.r Fri Sep 14 12:22:13 2018 -0400 @@ -0,0 +1,1004 @@ +#*************************************************************************************************************************************** +# Functions: Start +#*************************************************************************************************************************************** + +#=============================================================================== +# PCA +#=============================================================================== +multisample_PCA = function(df, sampleinfo_df, outfile) +{ + tempdf = df[,-1]; + tempcol = colnames(tempdf); + tempgrp = sampleinfo_df[tempcol,2]; + tempdf = t(tempdf) %>% as.data.frame(); + tempdf[is.na(tempdf)] = 0; + tempdf$Group = tempgrp; + png(outfile, width = 6, height = 6, units = 'in', res=300); + # bitmap(outfile, "png16m"); + g = autoplot(prcomp(select(tempdf, -Group)), data = tempdf, colour = 'Group', size=3); + plot(g); + dev.off(); +} + +#=============================================================================== +# Regression and Cook's distance +#=============================================================================== +singlesample_regression = function(PE_TE_data,htmloutfile, append=TRUE) +{ + rownames(PE_TE_data) = PE_TE_data$PE_ID; + regmodel = lm(PE_abundance~TE_abundance, data=PE_TE_data); + regmodel_summary = summary(regmodel); + + cat("

Linear Regression model fit between Proteome and Transcriptome data

\n", + "

Assuming a linear relationship between Proteome and Transcriptome data, we here fit a linear regression model.

\n", + '\n', + file = htmloutfile, append = TRUE); + + cat("\n", + "","\n", + "\n", + "\n", + "","\n", + "\n", + "\n", + "\n", + "\n", + file = htmloutfile, append = TRUE); + + cat("
ParameterValue
Formula","PE_abundance~TE_abundance","
Coefficients
",names(regmodel$coefficients[1]),"",regmodel$coefficients[1]," (Pvalue:", regmodel_summary$coefficients[1,4],")","
",names(regmodel$coefficients[2]),"",regmodel$coefficients[2]," (Pvalue:", regmodel_summary$coefficients[2,4],")","
Model parameters
Residual standard error",regmodel_summary$sigma," (",regmodel_summary$df[2]," degree of freedom)
F-statistic",regmodel_summary$fstatistic[1]," ( on ",regmodel_summary$fstatistic[2]," and ",regmodel_summary$fstatistic[3]," degree of freedom)
R-squared",regmodel_summary$r.squared,"
Adjusted R-squared",regmodel_summary$adj.r.squared,"
\n", file = htmloutfile, append = TRUE); + + cat( + "

Regression and diagnostics plots

\n", + file = htmloutfile, append = TRUE); + + outplot = paste(outdir,"/PE_TE_lm_1.png",sep="",collapse=""); + png(outplot, width = 10, height = 10, units = 'in',res=300); + # bitmap(outplot, "png16m"); + par(mfrow=c(1,1)); + plot(regmodel, 1, cex.lab=1.5); + dev.off(); + + outplot = paste(outdir,"/PE_TE_lm_2.png",sep="",collapse=""); + png(outplot,width = 10, height = 10, units = 'in', res=300); + # bitmap(outplot, "png16m"); + par(mfrow=c(1,1)); + plot(regmodel, 2, cex.lab=1.5); + dev.off(); + + outplot = paste(outdir,"/PE_TE_lm_5.png",sep="",collapse=""); + png(outplot, width = 10, height = 10, units = 'in',res=300); + # bitmap(outplot, "png16m"); + par(mfrow=c(1,1)); + plot(regmodel, 5, cex.lab=1.5); + dev.off(); + + cat('', file = htmloutfile, append = TRUE); + + cat( + '\n", + '\n', + file = htmloutfile, append = TRUE); + + cat( + '\n', + file = htmloutfile, append = TRUE); + + cat( + '\n', + '
', "

1) Residuals vs Fitted plot

2) Normal Q-Q plot of residuals

This plot checks for linear relationship assumptions.
If a horizontal line is observed without any distinct patterns, it indicates a linear relationship.
This plot checks whether residuals are normally distributed or not.
It is good if the residuals points follow the straight dashed line i.e., do not deviate much from dashed line.
\n', + file = htmloutfile, append = TRUE); + + + #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ + # Residuals data + #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ + res_all = regmodel$residuals; + res_mean = mean(res_all); + res_sd = sd(res_all); + res_diff = (res_all-res_mean); + res_zscore = res_diff/res_sd; + # res_outliers = res_all[which((res_zscore > 2)|(res_zscore < -2))] + + + tempind = which((res_zscore > 2)|(res_zscore < -2)); + res_PE_TE_data_no_outlier = PE_TE_data[-tempind,]; + res_PE_TE_data_no_outlier$residuals = res_all[-tempind]; + res_PE_TE_data_outlier = PE_TE_data[tempind,]; + res_PE_TE_data_outlier$residuals = res_all[tempind]; + + # Save the complete table for download (influential_observations) + temp_outlier_data = data.frame(res_PE_TE_data_outlier$PE_ID, res_PE_TE_data_outlier$TE_abundance, res_PE_TE_data_outlier$PE_abundance, res_PE_TE_data_outlier$residuals) + colnames(temp_outlier_data) = c("Gene", "Transcript abundance", "Protein abundance", "Residual value") + outdatafile = paste(outdir,"/PE_TE_outliers_residuals.txt", sep="", collapse=""); + write.table(temp_outlier_data, file=outdatafile, row.names=F, sep="\t", quote=F); + + + # Save the complete table for download (non influential_observations) + temp_all_data = data.frame(PE_TE_data$PE_ID, PE_TE_data$TE_abundance, PE_TE_data$PE_abundance, res_all) + colnames(temp_all_data) = c("Gene", "Transcript abundance", "Protein abundance", "Residual value") + outdatafile = paste(outdir,"/PE_TE_abundance_residuals.txt", sep="", collapse=""); + write.table(temp_all_data, file=outdatafile, row.names=F, sep="\t", quote=F); + + + cat('

Outliers based on the residuals from regression analysis

\n', + file = htmloutfile, append = TRUE); + cat('\n', + '\n', + '\n', + file = htmloutfile, append = TRUE); + + cat("\n", + "\n", + '\n', + '\n', + "
Residuals from Regression
ParameterValue
Mean Residual value",res_mean,"
Standard deviation (Residuals)",res_sd,"
Total outliers (Residual value > 2 standard deviation from the mean)',length(tempind),' (Download these ',length(tempind),' data points with high residual values here)
(Download the complete residuals data here)


\n", + file = htmloutfile, append = TRUE); + + #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ + + + cat('

', file = htmloutfile, append = TRUE); + + cat( + '\n', + file = htmloutfile, append = TRUE); + + cat( + '\n', + file = htmloutfile, append = TRUE); + + cat( + '

3) Residuals vs Leverage plot

This plot is useful to identify any influential cases, that is outliers or extreme values.
They might influence the regression results upon inclusion or exclusion from the analysis.

\n', + file = htmloutfile, append = TRUE); + + + + #^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ + # Cook's Distance + #^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ + cat('

INFLUENTIAL OBSERVATIONS

\n', + file = htmloutfile, append = TRUE); + cat( + '

Cook\'s distance computes the influence of each data point/observation on the predicted outcome. i.e. this measures how much the observation is influencing the fitted values.
In general use, those observations that have a Cook\'s distance > than ', cookdist_upper_cutoff,' times the mean may be classified as influential.

\n', + file = htmloutfile, append = TRUE); + + cooksd <- cooks.distance(regmodel); + + outplot = paste(outdir,"/PE_TE_lm_cooksd.png",sep="",collapse=""); + png(outplot, width = 10, height = 10, units = 'in', res=300); + # bitmap(outplot, "png16m"); + par(mfrow=c(1,1)); + plot(cooksd, main="Influential Obs. by Cook\'s distance", ylab="Cook\'s distance", xlab="Observations", type="n") # plot cooks distance + sel_outlier=which(cooksd>=as.numeric(cookdist_upper_cutoff)*mean(cooksd, na.rm=T)) + sel_nonoutlier=which(cooksdas.numeric(cookdist_upper_cutoff)*mean(cooksd, na.rm=T),names(cooksd),""), col="red", pos=2) # add labels + dev.off(); + + cat( + '', + '
In the above plot, observations above red line (',cookdist_upper_cutoff,' * mean Cook\'s distance) are influential. Genes that are outliers could be important. These observations influences the correlation values and regression coefficients

', + file = htmloutfile, append = TRUE); + + tempind = which(cooksd>as.numeric(cookdist_upper_cutoff)*mean(cooksd, na.rm=T)); + PE_TE_data_no_outlier = PE_TE_data[-tempind,]; + PE_TE_data_no_outlier$cooksd = cooksd[-tempind]; + PE_TE_data_outlier = PE_TE_data[tempind,]; + PE_TE_data_outlier$cooksd = cooksd[tempind]; + a = sort(PE_TE_data_outlier$cooksd, decreasing=T, index.return=T); + PE_TE_data_outlier_sorted = PE_TE_data_outlier[a$ix,]; + + cat( + '\n', + file = htmloutfile, append = TRUE); + + # Save the complete table for download (influential_observations) + temp_outlier_data = data.frame(PE_TE_data_outlier$PE_ID, PE_TE_data_outlier$TE_abundance, PE_TE_data_outlier$PE_abundance, PE_TE_data_outlier$cooksd) + colnames(temp_outlier_data) = c("Gene", "Transcript abundance", "Protein abundance", "Cook's distance") + outdatafile = paste(outdir,"/PE_TE_influential_observation.txt", sep="", collapse=""); + write.table(temp_outlier_data, file=outdatafile, row.names=F, sep="\t", quote=F); + + + # Save the complete table for download (non influential_observations) + temp_no_outlier_data = data.frame(PE_TE_data_no_outlier$PE_ID, PE_TE_data_no_outlier$TE_abundance, PE_TE_data_no_outlier$PE_abundance, PE_TE_data_no_outlier$cooksd) + colnames(temp_no_outlier_data) = c("Gene", "Transcript abundance", "Protein abundance", "Cook's distance") + outdatafile = paste(outdir,"/PE_TE_non_influential_observation.txt", sep="", collapse=""); + write.table(temp_no_outlier_data, file=outdatafile, row.names=F, sep="\t", quote=F); + + + cat("\n", + "\n", + + "
ParameterValue
Mean Cook\'s distance",mean(cooksd, na.rm=T),"
Total influential observations (Cook\'s distance > ",cookdist_upper_cutoff," * mean Cook\'s distance)",length(tempind),"
Observations with Cook\'s distance < ",cookdist_upper_cutoff," * mean Cook\'s distance",length(which(cooksd\n", + "


\n", + file = htmloutfile, append = TRUE); + + + #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ + # Scatter plot after removal of influential points + #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ + outplot = paste(outdir,"/AbundancePlot_scatter_without_outliers.png",sep="",collapse=""); + min_lim = min(c(PE_TE_data$PE_abundance,PE_TE_data$TE_abundance)); + max_lim = max(c(PE_TE_data$PE_abundance,PE_TE_data$TE_abundance)); + png(outplot, width = 10, height = 10, units = 'in', res=300); + # bitmap(outplot,"png16m"); + g = ggplot(PE_TE_data_no_outlier, aes(x=TE_abundance, y=PE_abundance))+geom_point() + geom_smooth() + xlab("Transcript abundance log fold-change") + ylab("Protein abundance log fold-change") + xlim(min_lim,max_lim) + ylim(min_lim,max_lim); + suppressMessages(plot(g)); + dev.off(); + + cat('\n', file = htmloutfile, append = TRUE); + # Before + cat("\n', file = htmloutfile, append = TRUE); + + # After + cat("\n', + file = htmloutfile, append = TRUE); + #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ + + + cor_result_pearson = cor.test(PE_TE_data_no_outlier[,"TE_abundance"], PE_TE_data_no_outlier[,"PE_abundance"], method = "pearson"); + cor_result_spearman = cor.test(PE_TE_data_no_outlier[,"TE_abundance"], PE_TE_data_no_outlier[,"PE_abundance"], method = "spearman"); + cor_result_kendall = cor.test(PE_TE_data_no_outlier[,"TE_abundance"], PE_TE_data_no_outlier[,"PE_abundance"], method = "kendall"); + + cat('\n', file = htmloutfile, append=TRUE); + + + cat('
Scatterplot: Before removalScatterplot: After removal
", '\n", + '
\n', file = htmloutfile, append=TRUE); + singlesample_cor(PE_TE_data, htmloutfile, append=TRUE); + cat('\n', + file = htmloutfile, append = TRUE); + + cat( + "\n", + "\n", + file = htmloutfile, append = TRUE) + cat("
ParameterMethod 1Method 2Method 3
Correlation method",cor_result_pearson$method,"",cor_result_spearman$method,"",cor_result_kendall$method,"
Correlation coefficient",cor_result_pearson$estimate,"",cor_result_spearman$estimate,"",cor_result_kendall$estimate,"
\n", file = htmloutfile, append = TRUE) + + + + if(dim(PE_TE_data_outlier)[1]<10) + { + tab_n_row = dim(PE_TE_data_outlier)[1]; + }else{ + tab_n_row = 10; + } + + cat("

Download the complete list of influential observations    ", + "Download the complete list (After removing influential points)
\n", + '

Top ',as.character(tab_n_row),' Influential observations (Cook\'s distance > ',cookdist_upper_cutoff,' * mean Cook\'s distance)

\n', + file = htmloutfile, append = TRUE); + + cat('\n', sep = "",file = htmloutfile, append = TRUE); + cat("\n", + file = htmloutfile, append = TRUE); + + + for(i in 1:tab_n_row) + { + cat( + '','\n', + '\n', + '\n', + '\n', + file = htmloutfile, append = TRUE); + } + cat('
GeneProtein Log Fold-ChangeTranscript Log Fold-ChangeCook's Distance
',as.character(PE_TE_data_outlier_sorted[i,1]),'',format(PE_TE_data_outlier_sorted[i,2], scientific=F),'',PE_TE_data_outlier_sorted[i,4],'',format(PE_TE_data_outlier_sorted[i,5], scientific=F),'


\n',file = htmloutfile, append = TRUE); + + +} + + + +#=============================================================================== +# Heatmap +#=============================================================================== +singlesample_heatmap=function(PE_TE_data, htmloutfile, hm_nclust){ + cat('
\n', + file = htmloutfile, append = TRUE); + + hc=hclust(dist(as.matrix(PE_TE_data[,c("PE_abundance","TE_abundance")]))) + hm_cluster = cutree(hc,k=hm_nclust); + + outplot = paste(outdir,"/PE_TE_heatmap.png",sep="",collapse=""); + png(outplot, width = 10, height = 10, units = 'in', res=300); + # bitmap(outplot, "png16m"); + par(mfrow=c(1,1)); + hmap = heatmap.2(as.matrix(PE_TE_data[,c("PE_abundance","TE_abundance")]), trace="none", cexCol=1, col=greenred(100),Colv=F, labCol=c("Proteins","Transcripts"), scale="col", hclustfun = hclust, distfun = dist); + dev.off(); + + cat('\n', + file = htmloutfile, append = TRUE); + + + temp_PE_TE_data = data.frame(PE_TE_data$PE_ID, PE_TE_data$TE_abundance, PE_TE_data$PE_abundance, hm_cluster); + colnames(temp_PE_TE_data) = c("Gene", "Transcript abundance", "Protein abundance", "Cluster (Hierarchical clustering)") + tempoutfile = paste(outdir,"/PE_TE_hc_clusterpoints.txt",sep="",collapse=""); + write.table(temp_PE_TE_data, file=tempoutfile, row.names=F, quote=F, sep="\t", eol="\n") + + + cat('
Heatmap of PE and TE abundance values (Hierarchical clustering)Number of clusters to extract: ',hm_nclust,'
Download the hierarchical cluster list
\n', + file = htmloutfile, append = TRUE); +} + + +#=============================================================================== +# K-means clustering +#=============================================================================== +singlesample_kmeans=function(PE_TE_data, htmloutfile, nclust){ + PE_TE_data_kdata = PE_TE_data; + k1 = kmeans(PE_TE_data_kdata[,c("PE_abundance","TE_abundance")], nclust); + outplot = paste(outdir,"/PE_TE_kmeans.png",sep="",collapse=""); + png(outplot, width = 10, height = 10, units = 'in', res=300); + # bitmap(outplot, "png16m"); + par(mfrow=c(1,1)); + scatter.smooth(PE_TE_data_kdata[,"TE_abundance"], PE_TE_data_kdata[,"PE_abundance"], xlab="Transcript Abundance", ylab="Protein Abundance", cex.lab=1.5); + legend(1, 95, legend=c("Cluster 1", "Line 2"), col="red", lty=1:1, cex=0.8) + legend(1, 95, legend="Cluster 2", col="green", lty=1:1, cex=0.8) + + + ind=which(k1$cluster==1); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="red", pch=16); + ind=which(k1$cluster==2); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="green", pch=16); + ind=which(k1$cluster==3); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="blue", pch=16); + ind=which(k1$cluster==4); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="cyan", pch=16); + ind=which(k1$cluster==5); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="black", pch=16); + ind=which(k1$cluster==6); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="brown", pch=16); + ind=which(k1$cluster==7); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="gold", pch=16); + ind=which(k1$cluster==8); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="thistle", pch=16); + ind=which(k1$cluster==9); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="yellow", pch=16); + ind=which(k1$cluster==10); + points(PE_TE_data_kdata[ind,"TE_abundance"], PE_TE_data_kdata[ind,"PE_abundance"], col="orange", pch=16); + dev.off(); + + cat('

\n', + file = htmloutfile, append = TRUE); + + tempind = order(k1$cluster); + tempoutfile = paste(outdir,"/PE_TE_kmeans_clusterpoints.txt",sep="",collapse=""); + write.table(data.frame(PE_TE_data_kdata[tempind, ], Cluster=k1$cluster[tempind]), file=tempoutfile, row.names=F, quote=F, sep="\t", eol="\n") + + + cat('\n', + file = htmloutfile, append = TRUE); + cat('
K-mean clusteringNumber of clusters: ',nclust,'
Download the cluster list


\n', + file = htmloutfile, append = TRUE); + +} + +#=============================================================================== +# scatter plot +#=============================================================================== +singlesample_scatter = function(PE_TE_data, outfile) +{ + min_lim = min(c(PE_TE_data$PE_abundance,PE_TE_data$TE_abundance)); + max_lim = max(c(PE_TE_data$PE_abundance,PE_TE_data$TE_abundance)); + png(outfile, width = 10, height = 10, units = 'in', res=300); + # bitmap(outfile, "png16m"); + g = ggplot(PE_TE_data, aes(x=TE_abundance, y=PE_abundance))+geom_point() + geom_smooth() + xlab("Transcript abundance log fold-change") + ylab("Protein abundance log fold-change") + xlim(min_lim,max_lim) + ylim(min_lim,max_lim); + suppressMessages(plot(g)); + # plot(g); + dev.off(); +} + +#=============================================================================== +# Correlation table +#=============================================================================== +singlesample_cor = function(PE_TE_data, htmloutfile, append=TRUE) +{ + cor_result_pearson = cor.test(PE_TE_data$TE_abundance, PE_TE_data$PE_abundance, method = "pearson"); + cor_result_spearman = cor.test(PE_TE_data$TE_abundance, PE_TE_data$PE_abundance, method = "spearman"); + cor_result_kendall = cor.test(PE_TE_data$TE_abundance, PE_TE_data$PE_abundance, method = "kendall"); + + cat( + '\n', + file = htmloutfile, append = TRUE); + + cat( + "\n", + "\n", + file = htmloutfile, append = TRUE) + cat("
ParameterMethod 1Method 2Method 3
Correlation method",cor_result_pearson$method,"",cor_result_spearman$method,"",cor_result_kendall$method,"
Correlation coefficient",cor_result_pearson$estimate,"",cor_result_spearman$estimate,"",cor_result_kendall$estimate,"
\n", file = htmloutfile, append = TRUE); + +} + +#=============================================================================== +# Boxplot +#=============================================================================== +multisample_boxplot = function(df, sampleinfo_df, outfile, fill_leg, user_xlab, user_ylab) +{ + tempdf = df[,-1, drop=FALSE]; + tempdf = t(tempdf) %>% as.data.frame(); + tempdf[is.na(tempdf)] = 0; + tempdf$Sample = rownames(tempdf); + tempdf1 = melt(tempdf, id.vars = "Sample"); + tempdf1$Group = sampleinfo_df[tempdf1$Sample,2]; + png(outplot, width = 6, height = 6, units = 'in', res=300); + # bitmap(outplot, "png16m"); + if(fill_leg=="Yes") + { + g = ggplot(tempdf1, aes(x=Sample, y=value, fill=Group)) + geom_boxplot() + labs(x=user_xlab) + labs(y=user_ylab) + }else{ + if(fill_leg=="No") + { + tempdf1$Group = c("case", "control") + g = ggplot(tempdf1, aes(x=Sample, y=value, fill=Group)) + geom_boxplot() + labs(x=user_xlab) + labs(y=user_ylab) + } + } + plot(g); + dev.off(); +} + + +#=============================================================================== +# Mean or Median of Replicates +#=============================================================================== + +mergeReplicates = function(TE_df,PE_df, sampleinfo_df, method) +{ +grps = unique(sampleinfo_df[,2]); + +TE_df_merged <<- sapply(grps, function(x){ + tempsample = sampleinfo_df[which(sampleinfo_df$Group==x),1] + if(length(tempsample)!=1){ + apply(TE_df[,tempsample],1,method); + }else{ + return(TE_df[,tempsample]); + } +}); +TE_df_merged <<- data.frame(as.character(TE_df[,1]), TE_df_merged); +colnames(TE_df_merged) = c(colnames(TE_df)[1], grps); + +PE_df_merged <<- sapply(grps, function(x){ + tempsample = sampleinfo_df[which(sampleinfo_df$Group==x),1] + if(length(tempsample)!=1){ + apply(PE_df[,tempsample],1,method); + }else{ + return(PE_df[,tempsample]); + } +}); + +PE_df_merged <<- data.frame(as.character(PE_df[,1]), PE_df_merged); +colnames(PE_df_merged) = c(colnames(PE_df)[1], grps); + +#sampleinfo_df_merged = data.frame(Sample = grps, Group = grps, stringsAsFactors = F); +sampleinfo_df_merged = data.frame(Sample = grps, Group = "Group", stringsAsFactors = F); + +return(list(TE_df_merged = TE_df_merged, PE_df_merged = PE_df_merged, sampleinfo_df_merged = sampleinfo_df_merged)); +} + +#=============================================================================== +# (T-Test or Wilcoxon ranksum test) and Volcano Plot +#=============================================================================== + +perform_Test_Volcano = function(TE_df_data,PE_df_data,TE_df_logfold, PE_df_logfold,sampleinfo_df, method, correction_method,volc_with) +{ + +PE_colnames = colnames(PE_df_data); +control_sample = sampleinfo_df[which(sampleinfo_df$Group=="control"),1]; +control_ind <<- sapply(control_sample, function(x){temp_ind = which(PE_colnames==x); as.numeric(temp_ind)}); +condition_sample = sampleinfo_df[which(sampleinfo_df$Group=="case"),1]; +condition_ind <<- sapply(condition_sample, function(x){temp_ind = which(PE_colnames==x); as.numeric(temp_ind)}); + +if(method=="mean"){ + #PE_pval = apply(PE_df_data[2:length(colnames(PE_df_data))],1,function(x) t.test(x[condition_ind-1], x[control_ind-1])$p.value); + PE_pval = apply(PE_df_data[2:length(colnames(PE_df_data))],1,function(x) {obj<-try(t.test(x[condition_ind-1], x[control_ind-1]),silent=TRUE); if(is(obj, "try-error")){return(NA)}else{return(obj$p.value)}}) +}else{ +if(method=="median"){ + PE_pval = apply(PE_df_data[2:length(colnames(PE_df_data))],1,function(x) {obj<-try(wilcox.test(x[condition_ind-1], x[control_ind-1]),silent=TRUE); if(is(obj, "try-error")){return(NA)}else{return(obj$p.value)}}) + # PE_pval = apply(PE_df_data[2:length(colnames(PE_df_data))],1,function(x) wilcox.test(x[condition_ind-1], x[control_ind-1])$p.value); + } +} +PE_adj_pval = p.adjust(PE_pval, method = correction_method, n = length(PE_pval)) + + +TE_colnames = colnames(TE_df_data); +control_sample = sampleinfo_df[which(sampleinfo_df$Group=="control"),1]; +control_ind <<- sapply(control_sample, function(x){temp_ind = which(TE_colnames==x); as.numeric(temp_ind)}); +condition_sample = sampleinfo_df[which(sampleinfo_df$Group=="case"),1]; +condition_ind <<- sapply(condition_sample, function(x){temp_ind = which(TE_colnames==x); as.numeric(temp_ind)}); + +if(method=="mean"){ + # TE_pval = apply(TE_df_data[2:length(colnames(TE_df_data))],1,function(x) t.test(x[condition_ind-1], x[control_ind-1])$p.value); + TE_pval = apply(TE_df_data[2:length(colnames(TE_df_data))],1,function(x) {obj<-try(t.test(x[condition_ind-1], x[control_ind-1]),silent=TRUE); if(is(obj, "try-error")){return(NA)}else{return(obj$p.value)}}) +}else{ + if(method=="median"){ + TE_pval = apply(TE_df_data[2:length(colnames(TE_df_data))],1,function(x) {obj<-try(wilcox.test(x[condition_ind-1], x[control_ind-1]),silent=TRUE); if(is(obj, "try-error")){return(NA)}else{return(obj$p.value)}}) + # TE_pval = apply(TE_df_data[2:length(colnames(TE_df_data))],1,function(x) wilcox.test(x[condition_ind-1], x[control_ind-1])$p.value); + } +} +TE_adj_pval = p.adjust(TE_pval, method = correction_method, n = length(TE_pval)) + + +PE_TE_logfold_pval = data.frame(TE_df_logfold$Gene, TE_df_logfold$LogFold, TE_pval, TE_adj_pval, PE_df_logfold$LogFold, PE_pval, PE_adj_pval); +colnames(PE_TE_logfold_pval) = c("Gene", "Transcript log fold-change", "p-value (transcript)", "adj p-value (transcript)", "Protein log fold-change", "p-value (protein)", "adj p-value (protein)"); +outdatafile = paste(outdir,"/PE_TE_logfold_pval.txt", sep="", collapse=""); +write.table(PE_TE_logfold_pval, file=outdatafile, row.names=F, sep="\t", quote=F); +cat("

Download the complete fold change data here
\n", + file = htmloutfile, append = TRUE); + + if(length(condition_ind)!=1) + { + # Volcano Plot + + if(volc_with=="adj_pval") + { + PE_pval = PE_adj_pval + TE_pval = TE_adj_pval + volc_ylab = "-log10 Adjusted p-value"; + }else{ + if(volc_with=="pval") + { + volc_ylab = "-log10 p-value"; + } + } + outplot = paste(outdir,"/PE_volcano.png",sep="",collapse=""); + png(outplot, width = 10, height = 10, units = 'in', res=300); + # bitmap(outplot, "png16m"); + par(mfrow=c(1,1)); + + plot(PE_df_logfold$LogFold, -log10(PE_pval), + xlab="log2 fold change", ylab=volc_ylab, + type="n") + sel <- which((PE_df_logfold$LogFold<=log(2,base=2))&(PE_df_logfold$LogFold>=log(0.5, base=2))) # or whatever you want to use + points(PE_df_logfold[sel,"LogFold"], -log10(PE_pval[sel]),col="black") + #sel <- which((PE_df_logfold$LogFold>log(2,base=2))&(PE_df_logfold$LogFoldlog(2,base=2))|(PE_df_logfold$LogFoldlog(2,base=2))|(PE_df_logfold$LogFold0.05) + sel=intersect(sel,sel1) + points(PE_df_logfold[sel,"LogFold"], -log10(PE_pval[sel]),col="blue") + abline(h = -log(0.05,base=10), col="red", lty=2) + abline(v = log(2,base=2), col="red", lty=2) + abline(v = log(0.5,base=2), col="red", lty=2) + dev.off(); + + + + outplot = paste(outdir,"/TE_volcano.png",sep="",collapse=""); + png(outplot, width = 10, height = 10, units = 'in', res=300); + # bitmap(outplot, "png16m"); + par(mfrow=c(1,1)); + + plot(TE_df_logfold$LogFold, -log10(TE_pval), + xlab="log2 fold change", ylab=volc_ylab, + type="n") + sel <- which((TE_df_logfold$LogFold<=log(2,base=2))&(TE_df_logfold$LogFold>=log(0.5, base=2))) # or whatever you want to use + points(TE_df_logfold[sel,"LogFold"], -log10(TE_pval[sel]),col="black") + #sel <- which((TE_df_logfold$LogFold>log(2,base=2))&(TE_df_logfold$LogFoldlog(2,base=2))|(TE_df_logfold$LogFoldlog(2,base=2))|(TE_df_logfold$LogFold0.05) + sel=intersect(sel,sel1) + points(TE_df_logfold[sel,"LogFold"], -log10(TE_pval[sel]),col="blue") + abline(h = -log(0.05,base=10), col="red", lty=2) + abline(v = log(2,base=2), col="red", lty=2) + abline(v = log(0.5,base=2), col="red", lty=2) + dev.off(); + + + + cat('
\n', file = htmloutfile, append = TRUE); + cat("\n', file = htmloutfile, append = TRUE); + cat("
Transcript Fold-ChangeProtein Fold-Change
", '", + '

\n', + file = htmloutfile, append = TRUE); + }else{ + cat('

!!! No replicates found. Cannot perform test to check significance of differential expression. Thus, no Volcano plot generated !!!

', + file = htmloutfile, append = TRUE); + } + +} + + +#*************************************************************************************************************************************** +# Functions: End +#*************************************************************************************************************************************** + + + + +#=============================================================================== +# Arguments +#=============================================================================== +noargs = 12; +args = commandArgs(trailingOnly = TRUE); +if(length(args) != noargs) +{ + stop(paste("Please check usage. Number of arguments is not equal to ",noargs,sep="",collapse="")); +} + +mode = args[1]; # "multiple" or "logfold" +method = args[2]; # "mean" or "median" +sampleinfo_file = args[3]; +proteome_file = args[4]; +transcriptome_file = args[5]; +correction_method = args[6]; +cookdist_upper_cutoff = args[7]; +numCluster = args[8]; +hm_nclust = args[9]; +volc_with = args[10]; + +htmloutfile = args[11]; # html output file +outdir = args[12]; # html supporting files + + +#=============================================================================== +# Check for file existance +#=============================================================================== +if(! file.exists(proteome_file)) +{ + stop(paste("Proteome Data file does not exists. Path given: ",proteome_file,sep="",collapse="")); +} +if(! file.exists(transcriptome_file)) +{ + stop(paste("Transcriptome Data file does not exists. Path given: ",transcriptome_file,sep="",collapse="")); +} + +#=============================================================================== +# Load library +#=============================================================================== +options(warn=-1); + +suppressPackageStartupMessages(library(dplyr)); +suppressPackageStartupMessages(library(data.table)); +suppressPackageStartupMessages(library(gplots)); +suppressPackageStartupMessages(library(ggplot2)); +suppressPackageStartupMessages(library(ggfortify)); + +#=============================================================================== +# Select mode and parse experiment design file +#=============================================================================== +if(mode=="multiple") +{ + expDesign = fread(sampleinfo_file, header = FALSE, stringsAsFactors = FALSE, sep="\t") %>% data.frame(); + expDesign_cc = expDesign[1:2,]; + + sampleinfo_df = expDesign[3:nrow(expDesign),]; + rownames(sampleinfo_df)=1:nrow(sampleinfo_df); + colnames(sampleinfo_df) = c("Sample","Group"); + + condition_cols = sampleinfo_df[which(sampleinfo_df[,2]==expDesign_cc[which(expDesign_cc[,1]=="case"),2]),1]; + condition_g_name = "case"; + control_cols = sampleinfo_df[which(sampleinfo_df[,2]==expDesign_cc[which(expDesign_cc[,1]=="control"),2]),1]; + control_g_name = "control"; + sampleinfo_df[which(sampleinfo_df[,2]==expDesign_cc[which(expDesign_cc[,1]=="case"),2]),2] = "case"; + sampleinfo_df[which(sampleinfo_df[,2]==expDesign_cc[which(expDesign_cc[,1]=="control"),2]),2] = "control"; + sampleinfo_df_orig = sampleinfo_df; +} + +if(mode=="logfold") +{ + sampleinfo_df = data.frame("Sample"= c("LogFold"), "Group"=c("Fold_Change")) +} + +#=============================================================================== +# Parse Transcriptome data +#=============================================================================== +TE_df_orig = fread(transcriptome_file, sep="\t", stringsAsFactor=F, header=T) %>% data.frame(); +if(mode=="multiple") +{ + TE_df = TE_df_orig[,c(colnames(TE_df_orig)[1],condition_cols,control_cols)]; +} +if(mode=="logfold") +{ + TE_df = TE_df_orig; + colnames(TE_df) = c("Genes", "LogFold"); +} +#=============================================================================== +# Parse Proteome data +#=============================================================================== +PE_df_orig = fread(proteome_file, sep="\t", stringsAsFactor=F, header=T) %>% data.frame(); +if(mode=="multiple") +{ + PE_df = PE_df_orig[,c(colnames(PE_df_orig)[1],condition_cols,control_cols)]; +} +if(mode=="logfold") +{ + PE_df = PE_df_orig; + colnames(PE_df) = c("Genes", "LogFold"); +} + +#============================================================================================================= +# Create directory structures and then set the working directory to output directory +#============================================================================================================= +if(! file.exists(outdir)) +{ + dir.create(outdir); +} +#=============================================================================== +# Write initial data summary in html outfile +#=============================================================================== + cat("\n", file = htmloutfile); + + cat("

QuanTP: Association between abundance ratios of transcript and protein


\n", + "

Input data summary

\n", + "
    \n", + "
  • Abbreviations used: PE (Proteome data) and TE (Transcriptome data)","

  • \n", + "
  • Input Proteome data dimension (Row Column): ", dim(PE_df)[1]," x ", dim(PE_df)[2],"
  • \n", + "
  • Input Transcriptome data dimension (Row Column): ", dim(TE_df)[1]," x ", dim(TE_df)[2],"

\n", + file = htmloutfile, append = TRUE); + + cat("

Table of Contents:

\n", + "
\n", + file = htmloutfile, append = TRUE); +#=============================================================================== +# Find common samples +#=============================================================================== +common_samples = intersect(sampleinfo_df[,1], colnames(TE_df)[-1]) %>% intersect(., colnames(PE_df)[-1]); + +if(length(common_samples)==0) +{ + stop("No common samples found "); + cat("Please check your experiment design file. Sample names (column names) in the Transcriptome and the Proteome data do not match. \n",file = htmloutfile, append = TRUE); +} + +#=============================================================================== +# Create subsets based on common samples +#=============================================================================== +TE_df = select(TE_df, 1, common_samples); +PE_df = select(PE_df, 1, common_samples); +sampleinfo_df = filter(sampleinfo_df, Sample %in% common_samples); +rownames(sampleinfo_df) = sampleinfo_df[,1]; + +#=============================================================================== +# Check for number of rows similarity +#=============================================================================== +if(nrow(TE_df) != nrow(PE_df)) +{ + stop("Number of rows in Transcriptome and Proteome data are not same i.e. they are not paired"); + cat("The correlation analysis expects paired TE and PE data i.e. (i)th gene/transcript of TE file should correspond to (i)th protein of PE file. In the current input provided there is mismatch in terms of number of rows of TE and PE file. Please make sure you provide paired data.\n",file = htmloutfile, append = TRUE); +} + +#=============================================================================== +# Number of groups +#=============================================================================== +ngrps = unique(sampleinfo_df[,2]) %>% length(); +grps = unique(sampleinfo_df[,2]); +names(grps) = grps; + +#=============================================================================== +# Change column1 name +#=============================================================================== +colnames(TE_df)[1] = "Gene"; +colnames(PE_df)[1] = "Protein"; + +#=============================================================================== +# Treat missing values +#=============================================================================== +TE_nacount = sum(is.na(TE_df)); +PE_nacount = sum(is.na(PE_df)); + +TE_df[is.na(TE_df)] = 0; +PE_df[is.na(PE_df)] = 0; + + +#=============================================================================== +# Decide based on analysis mode +#=============================================================================== +if(mode=="logfold") +{ + cat('

SAMPLE DISTRIBUTION

\n', + file = htmloutfile, append = TRUE); + + # TE Boxplot + outplot = paste(outdir,"/Box_TE.png",sep="",collape=""); + cat('\n', + '\n', + "\n', file = htmloutfile, append = TRUE); + multisample_boxplot(TE_df, sampleinfo_df, outplot, "Yes", "Samples", "Transcript Abundance data"); + + # PE Boxplot + outplot = paste(outdir,"/Box_PE.png",sep="",collape=""); + cat("
Boxplot: Transcriptome dataBoxplot: Proteome data
", '", '
\n', file = htmloutfile, append = TRUE); + multisample_boxplot(PE_df, sampleinfo_df, outplot, "Yes", "Samples", "Protein Abundance data"); + + cat('

CORRELATION

\n', + file = htmloutfile, append = TRUE); + + # TE PE scatter + outplot = paste(outdir,"/TE_PE_scatter.png",sep="",collape=""); + cat('\n', file = htmloutfile, append = TRUE); + cat("\n', file = htmloutfile, append = TRUE); + PE_TE_data = data.frame(PE_df, TE_df); + colnames(PE_TE_data) = c("PE_ID","PE_abundance","TE_ID","TE_abundance"); + singlesample_scatter(PE_TE_data, outplot); + + # TE PE Cor + cat("
Scatter plot between Proteome and Transcriptome Abundance
", '
", file = htmloutfile, append = TRUE); + singlesample_cor(PE_TE_data, htmloutfile, append=TRUE); + cat('*Note that correlation is sensitive to outliers in the data. So it is important to analyze outliers/influential observations in the data.
Below we use Cook\'s distance based approach to identify such influential observations.
\n', + file = htmloutfile, append = TRUE); + cat('
', + file = htmloutfile, append = TRUE); + + cat('

REGRESSION ANALYSIS

\n', + file = htmloutfile, append = TRUE); + + # TE PE Regression + singlesample_regression(PE_TE_data,htmloutfile, append=TRUE); + + cat('

CLUSTER ANALYSIS

\n', + file = htmloutfile, append = TRUE); + + # TE PE Heatmap + singlesample_heatmap(PE_TE_data, htmloutfile, hm_nclust); + + + # TE PE Clustering (kmeans) + singlesample_kmeans(PE_TE_data, htmloutfile, nclust=as.numeric(numCluster)) + +}else{ + if(mode=="multiple") + { + cat('

SAMPLE DISTRIBUTION

\n', + file = htmloutfile, append = TRUE); + + # TE Boxplot + outplot = paste(outdir,"/Box_TE_all_rep.png",sep="",collape=""); + cat('\n', + '\n', + "\n', file = htmloutfile, append = TRUE); + temp_df_te_data = data.frame(TE_df[,1], log(TE_df[,2:length(TE_df)])); + colnames(temp_df_te_data) = colnames(TE_df); + multisample_boxplot(temp_df_te_data, sampleinfo_df, outplot, "Yes", "Samples", "Transcript Abundance (log)"); + + # PE Boxplot + outplot = paste(outdir,"/Box_PE_all_rep.png",sep="",collape=""); + cat("
Boxplot: Transcriptome dataBoxplot: Proteome data
", '", '
\n', file = htmloutfile, append = TRUE); + temp_df_pe_data = data.frame(PE_df[,1], log(PE_df[,2:length(PE_df)])); + colnames(temp_df_pe_data) = colnames(PE_df); + multisample_boxplot(temp_df_pe_data, sampleinfo_df, outplot, "Yes", "Samples", "Protein Abundance (log)"); + + # Calc TE PCA + outplot = paste(outdir,"/PCA_TE_all_rep.png",sep="",collape=""); + multisample_PCA(TE_df, sampleinfo_df, outplot); + + # Calc PE PCA + outplot = paste(outdir,"/PCA_PE_all_rep.png",sep="",collape=""); + multisample_PCA(PE_df, sampleinfo_df, outplot); + + + # Replicate mode + templist = mergeReplicates(TE_df,PE_df, sampleinfo_df, method); + TE_df = templist$TE_df_merged; + PE_df = templist$PE_df_merged; + sampleinfo_df = templist$sampleinfo_df_merged; + rownames(sampleinfo_df) = sampleinfo_df[,1]; + + # TE Boxplot + outplot = paste(outdir,"/Box_TE_rep.png",sep="",collape=""); + cat('

Sample wise distribution (Box plot) after using ',method,' on replicates

\n', + "\n', file = htmloutfile, append = TRUE); + temp_df_te_data = data.frame(TE_df[,1], log(TE_df[,2:length(TE_df)])); + colnames(temp_df_te_data) = colnames(TE_df); + multisample_boxplot(temp_df_te_data, sampleinfo_df, outplot, "No", "Sample Groups", "Mean Transcript Abundance (log)"); + + # PE Boxplot + outplot = paste(outdir,"/Box_PE_rep.png",sep="",collape=""); + cat("
Boxplot: Transcriptome dataBoxplot: Proteome data
", '", '
\n', file = htmloutfile, append = TRUE); + temp_df_pe_data = data.frame(PE_df[,1], log(PE_df[,2:length(PE_df)])); + colnames(temp_df_pe_data) = colnames(PE_df); + multisample_boxplot(temp_df_pe_data, sampleinfo_df, outplot, "No", "Sample Groups", "Mean Protein Abundance (log)"); + + #=============================================================================== + # Calculating log fold change and running the "single" code part + #=============================================================================== + + TE_df = data.frame("Genes"=TE_df[,1], "LogFold"=apply(TE_df[,c(which(colnames(TE_df)==condition_g_name),which(colnames(TE_df)==control_g_name))],1,function(x) log(x[1]/x[2],base=2))); + PE_df = data.frame("Genes"=PE_df[,1], "LogFold"=apply(PE_df[,c(which(colnames(PE_df)==condition_g_name),which(colnames(PE_df)==control_g_name))],1,function(x) log(x[1]/x[2],base=2))); + + #=============================================================================== + # Treat missing values + #=============================================================================== + + TE_df[is.infinite(TE_df[,2]),2] = NA; + PE_df[is.infinite(PE_df[,2]),2] = NA; + TE_df[is.na(TE_df)] = 0; + PE_df[is.na(PE_df)] = 0; + + sampleinfo_df = data.frame("Sample"= c("LogFold"), "Group"=c("Fold_Change")) + #=============================================================================== + # Find common samples + #=============================================================================== + + common_samples = intersect(sampleinfo_df[,1], colnames(TE_df)[-1]) %>% intersect(., colnames(PE_df)[-1]); + TE_df = select(TE_df, 1, common_samples); + PE_df = select(PE_df, 1, common_samples); + sampleinfo_df = filter(sampleinfo_df, Sample %in% common_samples); + rownames(sampleinfo_df) = sampleinfo_df[,1]; + + # TE Boxplot + outplot = paste(outdir,"/Box_TE.png",sep="",collape=""); + cat('

Distribution (Box plot) of log fold change

', file = htmloutfile, append = TRUE); + cat('\n', + "\n', file = htmloutfile, append = TRUE); + multisample_boxplot(TE_df, sampleinfo_df, outplot, "Yes", "Sample (log2(case/control))", "Transcript Abundance fold-change (log2)"); + + # PE Boxplot + outplot = paste(outdir,"/Box_PE.png",sep="",collape=""); + cat("
Boxplot: Transcriptome dataBoxplot: Proteome data
", '", '
\n', file = htmloutfile, append = TRUE); + multisample_boxplot(PE_df, sampleinfo_df, outplot, "Yes", "Sample (log2(case/control))", "Protein Abundance fold-change(log2)"); + + + # Log Fold Data + perform_Test_Volcano(TE_df_orig,PE_df_orig,TE_df, PE_df,sampleinfo_df_orig,method,correction_method,volc_with) + + + + # Print PCA + + cat('

\n', + "\n', + "
PCA plot: Transcriptome dataPCA plot: Proteome data
", '", '
\n', + file = htmloutfile, append = TRUE); + + + + cat('

CORRELATION

\n', + file = htmloutfile, append = TRUE); + + # TE PE scatter + outplot = paste(outdir,"/TE_PE_scatter.png",sep="",collape=""); + cat('
\n', file = htmloutfile, append = TRUE); + cat("\n', file = htmloutfile, append = TRUE); + PE_TE_data = data.frame(PE_df, TE_df); + colnames(PE_TE_data) = c("PE_ID","PE_abundance","TE_ID","TE_abundance"); + singlesample_scatter(PE_TE_data, outplot); + + # TE PE Cor + cat("
Scatter plot between Proteome and Transcriptome Abundance
", '
\n", file = htmloutfile, append = TRUE); + singlesample_cor(PE_TE_data, htmloutfile, append=TRUE); + cat('*Note that correlation is sensitive to outliers in the data. So it is important to analyze outliers/influential observations in the data.
Below we use Cook\'s distance based approach to identify such influential observations.
\n', + file = htmloutfile, append = TRUE); + cat('
', + file = htmloutfile, append = TRUE); + + cat('

REGRESSION ANALYSIS

\n', + file = htmloutfile, append = TRUE); + + # TE PE Regression + singlesample_regression(PE_TE_data,htmloutfile, append=TRUE); + + cat('

CLUSTER ANALYSIS

\n', + file = htmloutfile, append = TRUE); + + #TE PE Heatmap + singlesample_heatmap(PE_TE_data, htmloutfile, hm_nclust); + + #TE PE Clustering (kmeans) + singlesample_kmeans(PE_TE_data, htmloutfile, nclust=as.numeric(numCluster)) + + } +} +cat("

Go To:

\n", + "\n", + "
TOP", + file = htmloutfile, append = TRUE); +cat("\n", file = htmloutfile, append = TRUE); diff -r 000000000000 -r f1db758949f4 quantp.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/quantp.xml Fri Sep 14 12:22:13 2018 -0400 @@ -0,0 +1,195 @@ + + Correlation between protein and transcript abundances + + r-data.table + r-gplots + r-dplyr + r-ggplot2 + r-ggfortify + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +@misc{QuanTP: A software resource for quantitative proteo-transcriptomic comparative data analysis and informatics, + author={Praveen Kumar, Priyabrata Panigrahi, James Johnson, Wanda Weber, Subina Mehta, Ray Sajulga, Caleb Easterly, Brian Crooker, Mohammad Heydarian, Krishanpal Anamika, Timothy Griffin, and Pratik Jagtap}, + year={2018}, + title={QuanTP} +} + + + diff -r 000000000000 -r f1db758949f4 test-data/exp_design_file.tabular --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/exp_design_file.tabular Fri Sep 14 12:22:13 2018 -0400 @@ -0,0 +1,6 @@ +case G1 +control G2 +D03_01 G1 +D03_02 G1 +M14_01 G2 +M14_02 G2 diff -r 000000000000 -r f1db758949f4 test-data/output.html --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/output.html Fri Sep 14 12:22:13 2018 -0400 @@ -0,0 +1,155 @@ + +

QuanTP: Association between abundance ratios of transcript and protein


+

Input data summary

+
    +
  • Abbreviations used: PE (Proteome data) and TE (Transcriptome data)

  • +
  • Input Proteome data dimension (Row Column): 2817 x 5
  • +
  • Input Transcriptome data dimension (Row Column): 2817 x 5

+

Table of Contents:

+
+

SAMPLE DISTRIBUTION

+ + + +
Boxplot: Transcriptome dataBoxplot: Proteome data
+

Sample wise distribution (Box plot) after using mean on replicates

+ +
Boxplot: Transcriptome dataBoxplot: Proteome data
+

Distribution (Box plot) of log fold change

+ +
Boxplot: Transcriptome dataBoxplot: Proteome data
+

Download the complete fold change data here
+
+ +
Transcript Fold-ChangeProtein Fold-Change

+

+ +
PCA plot: Transcriptome dataPCA plot: Proteome data
+

CORRELATION

+
+ +
Scatter plot between Proteome and Transcriptome Abundance
+ + + +
ParameterMethod 1Method 2Method 3
Correlation method Pearson's product-moment correlation Spearman's rank correlation rho Kendall's rank correlation tau
Correlation coefficient 0.1173569 0.1608612 0.1093701
+*Note that correlation is sensitive to outliers in the data. So it is important to analyze outliers/influential observations in the data.
Below we use Cook's distance based approach to identify such influential observations.
+

REGRESSION ANALYSIS

+

Linear Regression model fit between Proteome and Transcriptome data

+

Assuming a linear relationship between Proteome and Transcriptome data, we here fit a linear regression model.

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ParameterValue
Formula PE_abundance~TE_abundance
Coefficients
(Intercept) -0.06910598 (Pvalue: 1.220723e-05 )
TE_abundance 0.1712395 (Pvalue: 4.168015e-10 )
Model parameters
Residual standard error 0.8363295 ( 2815 degree of freedom)
F-statistic 39.31142 ( on 1 and 2815 degree of freedom)
R-squared 0.01377265
Adjusted R-squared 0.0134223
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Regression and diagnostics plots

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1) Residuals vs Fitted plot

2) Normal Q-Q plot of residuals

This plot checks for linear relationship assumptions.
If a horizontal line is observed without any distinct patterns, it indicates a linear relationship.
This plot checks whether residuals are normally distributed or not.
It is good if the residuals points follow the straight dashed line i.e., do not deviate much from dashed line.
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Outliers based on the residuals from regression analysis

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Residuals from Regression
ParameterValue
Mean Residual value 1.942328e-17
Standard deviation (Residuals) 0.836181
Total outliers (Residual value > 2 standard deviation from the mean) 164 (Download these 164 data points with high residual values here)
(Download the complete residuals data here)


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3) Residuals vs Leverage plot

This plot is useful to identify any influential cases, that is outliers or extreme values.
They might influence the regression results upon inclusion or exclusion from the analysis.

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INFLUENTIAL OBSERVATIONS

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Cook's distance computes the influence of each data point/observation on the predicted outcome. i.e. this measures how much the observation is influencing the fitted values.
In general use, those observations that have a Cook's distance > than 4 times the mean may be classified as influential.

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In the above plot, observations above red line ( 4 * mean Cook's distance) are influential. Genes that are outliers could be important. These observations influences the correlation values and regression coefficients

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ParameterValue
Mean Cook's distance 0.0004875011
Total influential observations (Cook's distance > 4 * mean Cook's distance) 115
Observations with Cook's distance < 4 * mean Cook's distance 2702


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Scatterplot: Before removalScatterplot: After removal
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ParameterMethod 1Method 2Method 3
Correlation method Pearson's product-moment correlation Spearman's rank correlation rho Kendall's rank correlation tau
Correlation coefficient 0.1173569 0.1608612 0.1093701
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ParameterMethod 1Method 2Method 3
Correlation method Pearson's product-moment correlation Spearman's rank correlation rho Kendall's rank correlation tau
Correlation coefficient 0.1334038 0.1611936 0.1082761
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Download the complete list of influential observations     Download the complete list (After removing influential points)
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Top 10 Influential observations (Cook's distance > 4 * mean Cook's distance)

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GeneProtein Log Fold-ChangeTranscript Log Fold-ChangeCook's Distance
CATHL2 -1.960863 4.88565 0.1432189
CD177 -4.173263 2.057499 0.06826605
CATHL1 -0.9912973 4.835209 0.05767091
HP 2.570727 3.885549 0.04680496
AZU1 -2.226356 -5.561874 0.03737565
ELANE -2.732479 -2.914936 0.03266198
PYGM -0.06079228 6.071712 0.03242859
LTF -2.4294 2.129742 0.02725017
ATP1A2 0.2871971 6.446299 0.01939256
C13H20orf194 -5.640732 -0.6697401 0.01852927


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CLUSTER ANALYSIS

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Heatmap of PE and TE abundance values (Hierarchical clustering)Number of clusters to extract: 5
Download the hierarchical cluster list
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K-mean clusteringNumber of clusters: 4
Download the cluster list


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Go To:

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TOP diff -r 000000000000 -r f1db758949f4 test-data/protein_data.tabular --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/protein_data.tabular Fri Sep 14 12:22:13 2018 -0400 @@ -0,0 +1,2818 @@ +Gene D03_01 D03_02 M14_01 M14_02 +ELANE 90604000.0 100390000.0 623380000.0 645960000.0 +AGL 976501500.0 960830000.0 697130000.0 719100000.0 +AGK 19878000.0 16422000.0 15565000.0 20390000.0 +HSPA6 67707000.0 176540000.0 117020000.0 121110000.0 +HSPA5 11009000000.0 6568200000.0 4795000000.0 5734100000.0 +HSPA4 117790000.0 30519000.0 90678000.0 87974000.0 +HSPA9 3544300000.0 3817500000.0 2235000000.0 2270700000.0 +HSPA8 7526200000.0 7289900000.0 3520000000.0 4052400000.0 +AGA 240460000.0 291780000.0 221830000.0 173620000.0 +LGALS3 186500000.0 258100000.0 242190000.0 301190000.0 +CSNK2A2 132720000.0 155250000.0 150050000.0 161600000.0 +PMM2 1632800000.0 1802500000.0 1614800000.0 1711000000.0 +PMM1 0.0 10979000.0 8686700.0 6732400.0 +ASS1 18134000000.0 18876000000.0 12966000000.0 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