Problem generating Violin Plot during QC

[CharlieYBart]

Hi,
I am a newbie working with scRNAseq data using Seurat5. I am currently processing 'filtered_feature_bc_matrix' dataset I've cellranger-ed. I've inputed:

obj_data <- Read10X("./outs/filtered_feature_bc_matrix")
obj <- CreateSeuratObject(obj_data, project = "toothgerm", min.cells = 3, min.features = 200)
obj[["percent.mt"]] <- PercentageFeatureSet(obj, pattern = "^mt-")
obj[["percent.Rp"]] <- PercentageFeatureSet(obj, pattern = '^Rp[ls]')
obj$log10GenesPerUMI <- log10(obj$nFeature_RNA / obj$nCount_RNA),

...which are very standard commands for QC and was working smoothly. However, when I tried to output violin plot for QC, using the following commands:

pdf("toothgerm_rawQC.pdf", width = 15, height = 4, title = "toothgerm_rawQC")
VlnPlot(obj, features = c("nFeature_RNA", "nCount_RNA",
"percent.mt","log10GenesPerUMI", "percent.Rp"), ncol = 5,layer="counts")
dev.off()

R returned:

Warning: Default search for "data" layer in "RNA" assay yielded no results; utilizing "counts" layer instead. Error in deparse(substitute(e2, env = caller_env(2))) : cannot get a slot ("slots") from an object of type "NULL"

I run:

obj_data <- Read10X("../2.runCR/toothgerm_e10-5/outs/filtered_feature_bc_matrix")
str(obj_data)
class(obj_data)

for data check, and R returned:

Formal class 'dgCMatrix' [package "Matrix"] with 6 slots ..@ i : int [1:27843821] 8 52 63 96 118 131 167 206 219 234 ... ..@ p : int [1:8912] 0 1137 4682 8529 12334 15993 19776 22440 26980 30001 ... ..@ Dim : int [1:2] 32285 8911 ..@ Dimnames:List of 2 .. ..$ : chr [1:32285] "Xkr4" "Gm1992" "Gm19938" "Gm37381" ... .. ..$ : chr [1:8911] "AAACCTGAGTAAGTAC-1" "AAACCTGCACAGTCGC-1" "AAACCTGCAGACGCAA-1" "AAACCTGCATAAGACA-1" ... ..@ x : num [1:27843821] 1 1 3 1 1 1 2 1 2 1 ... ..@ factors : list()
[1] "dgCMatrix"
attr(,"package")
[1] "Matrix"

Seems like the format and integrity of my data is ok?

So what's the problem and what should I do? Please help..

Thanks in advance...

[YoukaiFromAccounting]

Hello @CharlieYBart,

Thanks for reporting this issue. Would you mind posting your sessionInfo() to help us better understand your setup?

[CharlieYBart]

Hi @YoukaiFromAccounting,

Thank you so much for your reply. Yes, VlnPlot, alongside with all the other functions, are working well for the 'r4_cdd' environment.

Re your suggestion, do you mean detaching Seurat5 in 'r4_zyy' environment and then re-install the latest 'ggplot2', before plotting again?

Thanks again for your help:)

[CharlieYBart]

Hi @YoukaiFromAccounting ,

Updates: after re-installing Seurat package and ggplot2, it's working now.

Thanks a million!!

[CharlieYBart]

Hi @YoukaiFromAccounting ,

Sorry to bother you again - the same problem occurred! And this time it's on my local laptop. I was running the similar commands, and R returned the same error, please see below the screen shot:

My local R sessionInfo is:

What should I do? Last time I re-installed Seurat on the server and it seemed to work again, but here at my local laptop almost all packages are newly installed and up to date...

Thanks a lot. Look forward to your reply.

[YoukaiFromAccounting]

Hello @CharlieYBart,

Could you try attaching the ggplot2 package in your session then running your plotting code?

Via:

library(ggplot2)

#Check your ggplot2 version
packageVersion("ggplot2")
find.package("ggplot2")

[CharlieYBart]

Hi @YoukaiFromAccounting ,

Thanks so much for your reply. Problem solved - I think it's a package version issue. After re-installing Seurat 5.0, the codes can run without any error again.

Thanks again for your help.