cummeRbund csHeatmap column user-defined order

Question

I am using the R package cummeRbund (from Bioconductor) to visualize RNA-seq data, I created a cuffGeneSet instance called "DEG_genes" that contains 662 genes that are significantly differentially expressed between males and females. My goal is to create a heatmap using csHeatmap() in which the male and female samples (replicates) are separated but with a specific user-defined order within the sex category. I used:

> DEG<-diffData(genes(cuff))   # take differentially expressed genes
> DEG_significant<-subset(DEG,significant=='yes') # retain only significant changes
> DEG_sign_IDs <- DEG_significant$gene_id # retrieve IDs
> DEG_genes<-getGenes(cuff,DEG_sign_IDs) # get CuffGeneSet instance
> hmap<-csHeatmap(DEG_genes,clustering='none',labRow=F,replicates=T)

This gives me ALMOST what I want: the heatmap shows Females on the left and Males on the right but they are alphabetically ordered (Female_0,Female_1,Female_10,Female_11,Female_12...Female_19,Female_2,Female_20,Female_21..,Female_29 on the left and similarly for males Male_0,Male_1,Male_10...Male_19,Male_2,Male_20...etc on the right) and I want them to be in a specific order (clusterReps). I created a test vector with replicate names on a specific order (Males on the left with 0 and 6 echanged and females on the right) as follow:

clusterReps<-c("Male_6","Male_1","Male_2","Male_3","Male_4","Male_5","Male_0","Male_7","Male_8","Male_9","Male_10","Male_11","Male_12","Male_13","Male_14","Male_15","Male_16","Male_17","Male_18","Male_19","Male_20","Male_21","Male_22","Male_23","Male_24","Male_25","Male_26","Male_27","Male_28","Male_29","Male_30","Male_31","Male_32","Male_33","Female_0","Female_1","Female_2","Female_3","Female_4","Female_5","Female_6","Female_7","Female_8","Female_9","Female_10","Female_11","Female_12","Female_13","Female_14","Female_15","Female_16","Female_17","Female_18","Female_19","Female_20","Female_21","Female_22","Female_23","Female_24","Female_25","Female_26","Female_27","Female_28")

I would like the data to be exactly the same except the order of the columns that must follow the order of the "clusterReps" vector. Knowing that the heatmap is a ggplot, I looked everywhere for a solution the last 2 days but with no success (despite a closely ressembling problem with heatmap.2() instead of csHeatmap() on stackoverflow, I tried to get a replicate fpkm matrix and use heatmap.2 but could only use heatmap_2 and some options were not accepted). Using:

> hmap<-hmap+scale_x_discrete(limits=clusterReps)
Scale for 'x' is already present. Adding another scale for 'x', which will replace the existing scale.

only changes the x-axis labels but not the actual data (the heatmap remains identical). Is there a similar function that rearranges the columns and not just labels? Thanks in advance for your help, I'm not familiar with handling ggplot objects, and in particular heatmaps from cummeRbund.

EDIT: Here is what I can give as further information:

> DEG_genes
CuffGeneSet instance for  662  genes

Slots:
     annotation
     fpkm
     repFpkm
     diff
     count
     isoforms    CuffFeatureSet instance of size 930 
     TSS         CuffFeatureSet instance of size 785 
     CDS         CuffFeatureSet instance of size 230 
     promoters       CuffFeatureSet instance of size 662 
     splicing        CuffFeatureSet instance of size 785 
     relCDS      CuffFeatureSet instance of size 662 

> summary(DEG_genes)
     Length       Class        Mode 
        662 CuffGeneSet          S4 

I am afraid I can't give more information for the moment, please let me know if you want me to execute a command and report the output if it can help.

Answer 1

I am not very fluent in R, but I was having the same problem. To solve it I made a script that renames all my sample names in all the files inside the cuffdiff folder to something that will give the right order when sorted alphabetically, and then rebuild the database.