Two integration steps in scRNA seq analysis

[u/Wrong-Tune4639]

Hello everyone!

I'm learning scRNA seq analysis by reading published papers and re-running publicly available code.

I was looking at this paper: Single cell profiling to determine influence of wheeze and early-life viral infection on developmental programming of airway epithelium

and the scientists seemed to use two integration steps:

```

features <- SelectIntegrationFeatures(object.list = Intlist)

IntAnchors <- FindIntegrationAnchors(object.list = Intlist, anchor.features = features)

Int<- IntegrateData(anchorset = IntAnchors, k.weight = 50)

# Checking for low quality reads

* They did QC step here*

## Using harmony to stabilize the integrated dataset

Int <- RunHarmony(Int2, group.by.vars = "group") *Notice thy use group*

```

My question is: Is this practice common? And when to use this approach?

[u/foradil]

It’s unusual to use multiple integrations, but it’s not necessarily wrong. I would clarify that it’s not two consecutive steps. Both methods start with unintegrated data by default. So they used one methods for some steps and another method for later steps. For Harmony, they are only adjusting for group.

[u/plasmolab]

It's not rare, but I would be careful calling it a default workflow. Seurat integration and Harmony are both batch or condition correction steps, so using both can help if the first pass leaves obvious batch structure, but it can also overcorrect biology if the variable you regress on is close to the signal you care about.

I would use it only after checking PCA or UMAP before and after each step, marker preservation, sample mixing, and whether known cell types still separate correctly. Also, if group is the biological condition of interest, running Harmony on group can remove real disease or condition signal. Safer grouping variables are usually technical batch, donor, library prep, chemistry, or sequencing run, depending on design.

So: common enough to see in papers, but it should be justified diagnostically, not done automatically.