Question Regarding Differences Between VlnPlot2 and VlnPlot in SeuratExtend Package

[Sophia409]

Subject: Question Regarding Differences Between VlnPlot2 and VlnPlot in SeuratExtend Package

Dear Author of the SeuratExtend Package,

I am writing to seek clarification about the VlnPlot2 function in your SeuratExtend package. I have noticed that when using the same data and code, the output of VlnPlot2 differs significantly from the original VlnPlot function in Seurat.

Specifically, I executed the following code:

VlnPlot2(pbmc, features = genes, pt.size = 1, ncol = 1, split.by = "orig.ident") +  
VlnPlot(pbmc, features = genes, pt.size = 1, ncol = 1, split.by = "orig.ident")

The resulting plots from VlnPlot2 show many cells clustered around zero values, which is not observed in the original VlnPlot. Additionally, for genes such as CD14 and CD79A, many cells appear to have negative values in the VlnPlot2 output, which seems unusual.

Could you please help explain why these discrepancies occur? Is there a way to adjust VlnPlot2 so that its visualization aligns more closely with the original VlnPlot?

Thank you for your time and assistance. I look forward to your response.

Best regards,
Sophia

[huayc09]

Hi Sophia,

Thank you for reporting this issue with detailed screenshots!

The phenomenon you observed is caused by geom_jitter() applying random displacement in both horizontal AND vertical directions when there are many points with expression value of 0. These points are not truly negative values - they are artifacts from the vertical jittering behavior of geom_jitter().

Typically, this issue only occurs when there are a large number of cells with zero expression. For the pbmc example data, this shouldn't normally happen. In the tutorial, my plots appear normal without vertical displacement.

However, regardless of the circumstances, vertical jittering can be misleading and confusing. Thank you for bringing this to my attention! I have now enforced a restriction on vertical displacement to prevent this issue from occurring.

Please reinstall the package from GitHub:

remotes::install_github("huayc09/SeuratExtend")

After reinstalling, please test whether this fixes the problem. The points should now only jitter horizontally, with no vertical displacement.