issues with annotation

[troutena]

Hi All!

My name is Allison and I am a newb to analyzing single cell data. I am working through the Seurat and Signac vignettes that the Sajita lab provides but I am worried about an error code affecting downstream analysis.

For background, my data is from a 10x multiome run to give me scRNA and scATAC sequencing data. There are a few warnings that I'm worried may be affecting downstream commands. I basing my code of the pbmc vignette and my script is attached.
script.odt

Everything runs well until command:

annotations <- GetGRangesFromEnsDb(ensdb = EnsDb.Mmusculus.v79)

from which I get 21 warnings similar to this:

In .Seqinfo.mergexy(x, y) :
The 2 combined objects have no sequence levels in common. (Use
suppressWarnings() to suppress this warning.)

For background, I used refdata-cellranger-arc-mm10-2020-A-2.0.0 (provided on the 10x website) as my reference genome when running Cell Ranger ARC. Is this genome compatible with Ensembl genomes for annotation? I know there is a way to build a custom reference and then I could ensure compatibility. The annotations file that the code outputs looks normal, but I don't see any difference in the ATACob Seurat object when I try to annotate with the following code:

ATACob
granges(ATACob)
Annotation(object = ATACob) <- annotations
ATACob

Any help you could provide is very much appreciated. By the way I am using Seurat 4.0.4 and Signac 1.3.0 on R 4.1.0.

Thanks!

-Allison

[timoast]

Hi @troutena, those warnings can be ignored, I don't think there's any issue here

[troutena]

Hello @timoast thank you so much!

I think the error is in successfully annotating the Seurat object because later in the script when I run:

celltype.predictions <- TransferData(anchorset = transfer.anchors, refdata = GEXob$Annotations,
                                     weight.reduction = ATACob[["lsi"]], dims = 2:30)

I get the error:

Error: Cannot find 'Annotations' in this Seurat object

And when I perform the head function on GEXob, I don't see any annotation column. I see orig.ident, nCount_RNA, and nFeature_RNA. Do you think the annotation function isn't working?

Thanks!

-Allison

[timoast]

Hi Allison, this is not related to gene annotations stored in the ChromatinAssay object. Here GEXob$Annotations is referring to metadata associated with each cell in the GEXob object. The error indicates that there is no such metadata named "Annotations" in the Seurat object. I'd suggest looking over the code you're using to process the Seurat object and double check that the expected metadata is being added. If you have further questions, you can open an issue in the Seurat repository since this relates to Seurat functionality.