Is my pair (1 cancer and 1 normal) considered as biological replicates?

Question

[bioinforesearchquestions]

Hi,

I have been provided 30 samples (15 pairs). Each pair has one cancer and one normal sample. They are paired-end sequencing data for 15 cancer and 15 normal samples. I am doing RNA-seq analysis for these samples using DESeq package. I used HTSeq_count to count the number of reads for each gene.

Group1 - Sample1 is cancer and Sample2 is normal

Group2 - Sample3 is cancer and Sample4 is normal

Group3 - Sample5 is cancer and Sample6 is normal

... ...

Group15- Sample29 is cancer and Sample30 is normal

The technician asked me to compare each group (1cancer and 1normal) individually. So, I have to perform 15 pairwise comparisons.

1) Is my pair (1 cancer and 1 normal) considered as biological replicates?

Answer 1

[John]

They could not be less biological replicates :P

If each of the 15 normal samples were from the same individual and the same tissue, they would be considered biological replicates. For the cancers it's a bit of an academic moot point if cancerous samples can ever have true biological replicates. 15 biopsies from the same cancerous lump might contain 2 or 3 distinct populations of cancerous cells with their own phenotypic profile. But i'd be a lier if i said there were no publications where such comparisons where done and the samples called biological cancerous replicates.

Your 15 groups could be considered technical replicates if the exact same technique was performed on all of them - but usually with technical replicates it is assumed they are also biological replicates, so it depends on who you ask.

[bioinforesearchquestions]

The technician told me that each group (1 cancer and 1 normal) are from different patients.

So I have to perform differential gene expression using DESeq without replicates option. Am I correct?

[John]

Oh, hm, i have no idea. I presume all the cancers are the same kind of cancer phenotypically, but may or may not have the same underlying mechanism?

I'm sure there's a model for that in DESeq, but I don't know what it is. I suppose you could say your 15 groups are replicates, however I don't think that's fair.

Basically I don't know, you should ask someone who specifically knows how to use DESeq for cancer studies. I've only ever studied inherited cancers, looking at genomic variations, never looked at expression.