Hello,

I cannot seem to figure out why my heat map does not group my samples by condition when I use collapse replicate function. The only thing that is changed is dds --> ddsColl and vstdds --> vstddsColl. Any help would be appreciated. 

### DESeq2 Analysis on Personal ###

setwd('/Users/Corey/Desktop/DESeq2/Final_DESeq2/')

library('DESeq2')

cts <- read.csv(file='/Users/Corey/Desktop/DESeq2/Final_DESeq2/cts.csv')

nrow(cts)

ncol(cts)

colData <- read.csv(file='colData.csv')

ncol(colData)

nrow(colData)

rownames(cts) <- cts$Geneid

cts$Geneid <- NULL

#library("BiocParallel")

#register(MulticoreParam(12))

dds <- DESeqDataSetFromMatrix(countData=cts, colData=colData, design= ~ patient + condition)

keep <- rowSums(counts(dds) >= 10) >= 5

dds <- dds[keep,]

dds$condition <- relevel(dds$condition, ref='NT')

# micheal said it doesnt matter what the refernce is b/c the comparision between the sample remains the same

ddsColl <- collapseReplicates(dds, dds$id)

ddsColl <- DESeq(ddsColl)


# Does ddsColl throw off heatmap generation

dds <- DESeq(dds)

resultsNames(ddsColl)

res <- results(ddsColl, name="condition_MPT_vs_NT", lfcThreshold=0.585, alpha=0.05)

res

resOrder <- res[order(res$padj),]

write.csv(as.data.frame(resOrder), file='DESeqResults.csv')

summary(resOrder)

library('pheatmap')

#ddsColl

pdf('heatmap_top100.pdf', height=10, )
select <- order(rowMeans(counts(ddsColl,normalized=TRUE)), decreasing=TRUE)[1:100]
df <- as.data.frame(colData(ddsColl)[,c("condition","patient")])
pheatmap(assay(vstddsColl)[select,], cluster_rows=FALSE,show_rownames=FALSE,cluster_cols=FALSE, annotation_col=df)
dev.off()

#dds

pdf('heatmaptop100.pdf', height=10, )
select <- order(rowMeans(counts(dds,normalized=TRUE)), decreasing=TRUE)[1:100]
df <- as.data.frame(colData(dds)[,c("condition","patient")])
pheatmap(assay(vstdds)[select,], cluster_rows=FALSE,show_rownames=FALSE,cluster_cols=FALSE, annotation_col=df)
dev.off()

pheatmap groups samples by distance. Take a look at the help pages in the pheatmap package. I don’t understand how this has anything to do with collapseReplicates. These operations aren’t repeated. If you collapse technical replicates, pheatmap doesn’t “know” about this, it only sees the collapsed samples you provide to it.