DESeq2 on metagenome KO counts

Question

[young_bioinformatician]

Hi all,

I have metagenome data. I aggregate raw counts per KO × sample. I want to do differential abundance between two time groups using DESeq2. After that, I want to show abundance heatmaps and volcanot plot.

My first question is about DESeq analysis. Is it valid to use DESeq2 on KO-level metagenome counts? I don’t have a “true control,” so I plan to set one group as the reference and interpret log2FC relative to that.

Second, is it acceptable to plot a heatmap using VST-transformed values? Alternatively, I could take the top 50 significant KOs from DESeq2, extract their CPM values, and plot a CPM heatmap, but I expect the visual patterns to differ a bit because VST and CPM are different scales.

Thank you very much.

[Aleksandra]

(I apologise for my previous reply; I wanted to be as detailed as possible) DESeq2 handles KO counts perfectly well. For a time-series design without a separate control group, simply set the reference level in the design formula to the first time point. For the heatmap, definitely use the VST-transformed values. The patterns will differ from CPM and the VST is what you want. This stabilises the variance, ensuring that the clustering isn't skewed by the most abundant KOs.

[ATpoint]

Both vst or logcpm will favor expression level (magnitude of counts) rather than differences in a hclust/heatmap. For differences you need to scale counts first, be it vst or logcpm. See Scaling RNA-Seq data before clustering?

[Aleksandra]

Thank you very much. I mistakenly drew a superficial analogy. I equated KO counters with gene counters.

[Aleksandra]

I focused on the fact that DESeq2 models overdispersion, but overlooked its implicit assumption: normalisation works when the main source of variation between samples is sequencing depth, rather than systematic variation in feature length.

[ATpoint]

This statement makes zero sense in response to what I wrote.

[ATpoint]

The DESeq2 developer has advised many times against DESeq2 for metagenomics. Please search for related posts over at support.bioconductor.org where he advised for alternatives.

Answer 1

[andres.firrincieli]

I wouldn’t use DESeq2 on aggregated raw counts per KO, because doing so means accepting two pretty big assumptions:

1. All genes with the same KO have the same length, which usually isn’t true.
2. KOs make up a small part of the total coding sequence. When DESeq2 normalises for sequencing depth, it’s assuming that the proportion of CDS with a KOs stays roughly the same across samples. If that’s not the case, the normalisation might not work well.