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mouse+human cells in 10x scRNA-seq

Hi everyone,

I'm analyzing 10x scRNA-seq data generated from xenografts (mouse + human tissues). I have the following workflow to label cells as either mouse or human:

  1. Align 10x scRNA-seq data to mouse+human combined genome using cellranger count.
  2. Use the file generated by cellranger count (gem_classification.csv) which assigns mouse, human, or multiplet to each cell to classify each cell type.
  3. Realign the fastq files to the mouse genome ( we are interested in the mouse cells) using cellranger count
  4. Use the classification from Step 2 to filter out human cells in Seurat.

Would this be the right approach or is there a better alternative that you would recommend?

Thanks a lot!

cellranger 10x singlecell
• 4,071 views • link
updated by jared.andrews07 jared.andrews07 • written by Sy80 • Sy80

For the third step I get the error

Martian Runtime - v4.0.8
RuntimeError: pipestance 'HNP_01_P5_culture' already exists with different invocation file __HNP_01_P5_culture.mro

How should it be modified so that it doesn't baulk about the sample already being processed once?

• 0 views • link
written by dario.garvan dario.garvan

Change the ID. Or output in a different folder.

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written by jared.andrews07 jared.andrews07

1 answer

This approach is fine, but is there a reason you aren't just using the output from step 2 and limiting to the mouse cells/genes within it?

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written by jared.andrews07 jared.andrews07

If there are stray reads in "mouse" cells which aligned to human for some reason, reliagning to mouse alone will force those reads to be assigned to a mouse gene.

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written by swbarnes2 swbarnes2

I guess I don't have a good grasp on how much of a difference it'd make, would be interesting to know if there are specific genes where this is a particular issue.

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written by jared.andrews07 jared.andrews07

Thank you to both for your input. I also thought that alignment may improve if only a single ref genome is present, perhaps only for a small fraction of genes - though this is just an assumption. I may try both ways just to compare.

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written by Sy80 • Sy80

If you notice much of a difference, be sure to give us the update!

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written by jared.andrews07 jared.andrews07

Will do so!

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written by Sy80 • Sy80

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