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Combining technical repeat with original for a clogged multiome sample?

Hi all, I have a multiplexed 10x Multiome (snRNA + snATAC) dataset where one of the samples had a technical issue I'd like guidance on.

The original sample was clogged during loading and the Cell Ranger ARC report estimated only ~1,500 cells The experiment was repeated and recovered ~5,500 cells (a separate library / sequencing run) Other samples in my cohort have ~8,000–11,000 cells per pool The pool is multiplexed (2 donors per pool), demultiplexed via genotype using Vireo

Is it appropriate to combine the original (1,500-cell) and repeat (5,500-cell), or should I treat them as separate samples (or just use the repeat and discard the original)? If combining is OK:

Should the libraries be merged at the count matrix level (concatenate cells), or should I align fragments at the sample/donor level first? Are there batch-effect considerations between the two runs that I need to account for (e.g., the two were sequenced separately, possibly with different chemistry batch / loading conditions)?

I'm relatively new to single-cell analysis so any pointers to literature or worked examples would be hugely appreciated.

multiome single cell

1 answer

A clog doesn't just lose cells, it biases which cells you got - larger nuclei and debris block first, so your 1,500 are a compositionally skewed subset, and the extra time on the instrument means more ambient RNA and more stressed nuclei. So it depends on the analysis: if you're comparing cell type composition across donors, merging a biased 1,500 into a clean 5,500 corrupts the very thing you're measuring and I'd just discard the original. If you only need cells for annotation or per-cell-type expression, merging is fine.

If you do merge, do it at the count matrix level with run kept as a batch covariate, not by pooling fragments. Two libraries means two emulsions, two ambient pools and two sequencing batches, and pooling fragments as though it were one library hides all of that.

One trap specific to your setup: Vireo's donor0/donor1 labels are arbitrary per run unless you give it a reference genotype VCF. Donor0 in the repeat isn't necessarily the same person as donor0 in the original. Either anchor both runs to a genotype reference, or match them afterwards using the genotypes Vireo writes out - otherwise you can silently merge one donor's cells into the other's.

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