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Mitochondrial genes as Seurat markers in nuclear sequencing data

I've integrated some single nucleus RNA seq data after the standard Seurat preprocessing workflow. I normalized with SCTransform, regressed mitochondrial DNA, removed cells with >5% mitochondrial DNA (not in this order).

obj <-CreateSeuratObject(obj.data)
obj <- PercentageFeatureSet(object = obj, pattern = "^mt-", col.name = "percent.mt")
obj <- SCTransform(object = obj, vars.to.regress = "percent.mt", verbose = FALSE)

Then I integrated with FindIntegrationAnchors, etc.

When I identify markers between my treated and untreated data (using PrepSCTFindMarkers and then DESeq2 FindAllMarkers), I have mitochondrial genes come up as markers (~0.4 avg_log2FC). This is un-expected considering this is nuclear sequencing data, and I've regressed percent.mt.

Am I missing something? Did I do something incorrectly, or is there something wrong with the data, or is this totally normal?

Thanks!

snrnaseq seurat mitochondria

Did you also filter your "cells" based on mtRNA levels (remove cells with >X% mitochondrial reads)? I have no idea how the nucleus RNA-seq works but it could be that empty wells still contain extra-cellular mitochondria.

I did. I removed any cells with more than 5% mitochondrial DNA, but certain mitochondrial genes (ie mt-Nd3) are coming up as differentially expressed.

1 answer

Encountered the same issue, may I ask if theres any updates?

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