RNAseq and heatmap
Entering edit mode
Merlin ▴ 10
Last seen 20 months ago

Hi guys,

When at the end of the analysis I export the results with this code:


I have a file containing a list of genes with Log2FC and p-value. let say I have two conditions treated and untreated (6 and 2 ), are the values of gene expression of two replicates (untreated) normalized and compared with the values of the 6 replicates (treated) ? I mean are this taken as a single condition isn't it?

What if I have more conditions? let say 4 different conditions, to create that table 3 will be considered as a unique and one as reference condition?

Thank you

also, to make a heatmap where all the biological replicates cluster together I could take the top 1000 gene that are similar among replicates and different with the reference level but I don't know if that is right and/or DESeq2 can do that, what do you guys think?


deseq2 RNA-seq Heatmap • 907 views
Entering edit mode
Last seen 3 hours ago
United States

Take a look at the workflow which describes what shows up in the results table:


To contrast multiple groups, you need to call results() multiple times using the contrast argument. See:


For the last case, you can find these genes by performing a likelihood ratio test. If you have a dds with design ~condition, you can do:

dds <- DESeq(dds, test="LRT", reduced=~1)
res <- results(dds)
heatmap.genes <- head(order(res$padj), n)

where n is the number of genes for the heatmap.

Entering edit mode

Thank you, now the first part is clear enough,

For the heatmap,I tried this code as you say but I have this result,

pheatmap(heatmap.genes) Error in hclust(d, method = method) : must have n >= 2 objects to cluster

I think is missing the information of the samples name which in my previous command for heatmao was rld,

topGenes<- head(order(rowVars(assay(rld)), decreasing = TRUE),1000)

Can you tell me how to fix it?

I used this to show the heatmap

library("pheatmap") pheatmap(heatmap.genes)

thank you

Entering edit mode

Looks like you’re having some issues going from the transformer data to the heatmap. I’d recommend go one step at a time and make sure you have what you are expecting at each step. Eg heatmap.genes is not data, it’s a set of numeric indices. You should get in the habit of looking at the various objects to see what they represent, using eg head() and dim(), etc.


Login before adding your answer.

Traffic: 422 users visited in the last hour
Help About
Access RSS

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6