Single cell data

[DrSawal]

my data set is named as HS
when I am giving this command
HS <- FindVariableFeatures(HS, selection.method = "vst", nfeatures = 2000)

Identify the 10 most highly variable genes

top10 <- head(VariableFeatures(HS), 10)

plot variable features with and without labels

plot1 <- VariableFeaturePlot(HS)
plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE)
plot1 + plot2

I am getting this error in Seurat
Error in .SelectFeatures():
! None of the features provided are present in the feature set

Backtrace:
▆

1. ├─Seurat::FindVariableFeatures(object = HS)
2. └─Seurat:::FindVariableFeatures.Seurat(object = HS)
3. ├─Seurat::FindVariableFeatures(...)
4. └─Seurat:::FindVariableFeatures.StdAssay(...)

5. ├─SeuratObject::VariableFeatures(...)
   

6. └─SeuratObject:::VariableFeatures.Assay5(...)
   

7.   ├─SeuratObject::.SelectFeatures(...)
   

8.   └─SeuratObject:::.SelectFeatures.list(...)
   

Run rlang::last_trace(drop = FALSE) to see 1 hidden frame.

Can you please help me in resolving this issue

[DrSawal]

When I am giving this command
plot1 <- VariableFeaturePlot(HS)
plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE)
plot1 + plot2

I am getting this error
Error in HVFInfo():
! Please run either 'FindVariableFeatures' or 'SCTransform'
Run rlang::last_trace() to see where the error occurred.

[alexwskh]

Have you run FindVariableFeatures or SCTransform on your data?

[DrSawal]

I tried a couple of times. But couldn't get the results. Can you send me the exact command for that. My seurat object is HS

…

________________________________ From: alexwskh ***@***.***> Sent: Thursday, December 19, 2024 3:53 PM To: satijalab/seurat ***@***.***> Cc: Humaira Sawal ***@***.***>; Author ***@***.***> Subject: [EXTERNAL] Re: [satijalab/seurat] Single cell data (Issue #9561) Have you run FindVariableFeatures or SCTransform on your data? — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you authored the thread. Message ID: <satijalab/seurat/issues/9561/2555754361@ github. com> Have you run FindVariableFeatures or SCTransform on your data? — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/satijalab/seurat/issues/9561*issuecomment-2555754361__;Iw!!GobTDDpD7A!PS8r-1EXgc7P-ucHay-qlg13Y4vXxg1DIX8yvkdeY7GR3uoMTdTCbjZjxia1O4xOjVnfw5bwgpQATjuzGg0ag2539w$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/BN22OXA2JNC6Y3VOBSF7JIL2GMW4PAVCNFSM6AAAAABT5VBBAKVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDKNJVG42TIMZWGE__;!!GobTDDpD7A!PS8r-1EXgc7P-ucHay-qlg13Y4vXxg1DIX8yvkdeY7GR3uoMTdTCbjZjxia1O4xOjVnfw5bwgpQATjuzGg3wM7lneA$>. You are receiving this because you authored the thread.Message ID: ***@***.***>

[alexwskh]

It seems like you are unsure of your workflow and what you are trying to do. You should follow this tutorial... If you can complete that tutorial, you should understand where you are failing.

https://satijalab.org/seurat/articles/pbmc3k_tutorial

[DrSawal]

I am already using this tutorial for my work but I am stuck now because I am getting such types of errors. Error in `PrepDR5()`: ! No layer matching pattern 'scale.data' not found. Please run ScaleData and retry Run `rlang::last_trace()` to see where the error occurred.

rlang::last_trace()

<error/rlang_error> Error in `PrepDR5()`: ! No layer matching pattern 'scale.data' not found. Please run ScaleData and retry --- Backtrace: ▆ 1. ├─Seurat::RunPCA(HS) 2. └─Seurat:::RunPCA.Seurat(HS) 3. ├─Seurat::RunPCA(...) 4. └─Seurat:::RunPCA.StdAssay(...) 5. └─Seurat:::PrepDR5(object = object, features = features, layer = layer, verbose = verbose) Run rlang::last_trace(drop = FALSE) to see 1 hidden frame.

…

________________________________ From: alexwskh ***@***.***> Sent: Thursday, December 19, 2024 4:19 PM To: satijalab/seurat ***@***.***> Cc: Humaira Sawal ***@***.***>; Author ***@***.***> Subject: [EXTERNAL] Re: [satijalab/seurat] Single cell data (Issue #9561) It seems like you are unsure of your workflow and what you are trying to do. You should follow this tutorial. . . If you can complete that tutorial, you should understand where you are failing. https: //satijalab. org/seurat/articles/pbmc3k_tutorial It seems like you are unsure of your workflow and what you are trying to do. You should follow this tutorial... If you can complete that tutorial, you should understand where you are failing. https://satijalab.org/seurat/articles/pbmc3k_tutorial<https://urldefense.com/v3/__https://satijalab.org/seurat/articles/pbmc3k_tutorial__;!!GobTDDpD7A!NY4JNDz3cq38ut5LBBgGrYRDUST_UGUp1joNCFWpcFfbi6a1NmEvRnhcBuxgdvzDsUY-dmWo8C6XtSAJmTPFLGoQzA$> — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/satijalab/seurat/issues/9561*issuecomment-2555792597__;Iw!!GobTDDpD7A!NY4JNDz3cq38ut5LBBgGrYRDUST_UGUp1joNCFWpcFfbi6a1NmEvRnhcBuxgdvzDsUY-dmWo8C6XtSAJmTOF0BhKKA$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/BN22OXH5ZVRI3JPOO4N75HT2GMZ6LAVCNFSM6AAAAABT5VBBAKVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDKNJVG44TENJZG4__;!!GobTDDpD7A!NY4JNDz3cq38ut5LBBgGrYRDUST_UGUp1joNCFWpcFfbi6a1NmEvRnhcBuxgdvzDsUY-dmWo8C6XtSAJmTNhrFqoQg$>. You are receiving this because you authored the thread.Message ID: ***@***.***>

[alexwskh]

I mean it is telling you in the error message what to look for. Your object doesn't have any scale.data layers in it, and it is asking you to run ScaleData.

The tutorial is pretty clear. You import your object, you subset it based on mitochondrial content and counts, you run NormalizeData and then FindVariableFeatures before heading onto the next steps.

I would recommend you spend a bit more time understanding what each step is actually doing, and that you aren't jumping ahead. My best guess is that you are missing earlier required steps.

[DrSawal]

You are so much right. I am stuck here just to figure out which step I am missing in between.

…

________________________________ From: alexwskh ***@***.***> Sent: Thursday, December 19, 2024 4:32 PM To: satijalab/seurat ***@***.***> Cc: Humaira Sawal ***@***.***>; Author ***@***.***> Subject: [EXTERNAL] Re: [satijalab/seurat] Single cell data (Issue #9561) I mean it is telling you in the error message what to look for. Your object doesn't have any scale. data layers in it, and it is asking you to run ScaleData. The tutorial is pretty clear. You import your object, you subset it based on mitochondrial I mean it is telling you in the error message what to look for. Your object doesn't have any scale.data layers in it, and it is asking you to run ScaleData. The tutorial is pretty clear. You import your object, you subset it based on mitochondrial content and counts, you run NormalizeData and then FindVariableFeatures before heading onto the next steps. I would recommend you spend a bit more time understanding what each step is actually doing, and that you aren't jumping ahead. My best guess is that you are missing earlier required steps. — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/satijalab/seurat/issues/9561*issuecomment-2555808789__;Iw!!GobTDDpD7A!LnGSdGFS8ajlcWzh6eGiAm8PhscWC78DtCvYZvLCyp5nGT6i-A5nImWG9gnaZzsJsBbnDi_0HkXdun3siRZpemmm7g$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/BN22OXAGIP2TJISPWC7XDPT2GM3NNAVCNFSM6AAAAABT5VBBAKVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDKNJVHAYDQNZYHE__;!!GobTDDpD7A!LnGSdGFS8ajlcWzh6eGiAm8PhscWC78DtCvYZvLCyp5nGT6i-A5nImWG9gnaZzsJsBbnDi_0HkXdun3siRa_eYs3Ng$>. You are receiving this because you authored the thread.Message ID: ***@***.***>

[DrSawal]

which command ca be use for ScaleData in seurat?

…

________________________________ From: Humaira Sawal ***@***.***> Sent: Thursday, December 19, 2024 4:34 PM To: satijalab/seurat ***@***.***> Subject: Re: [EXTERNAL] Re: [satijalab/seurat] Single cell data (Issue #9561) You are so much right. I am stuck here just to figure out which step I am missing in between.

________________________________ From: alexwskh ***@***.***> Sent: Thursday, December 19, 2024 4:32 PM To: satijalab/seurat ***@***.***> Cc: Humaira Sawal ***@***.***>; Author ***@***.***> Subject: [EXTERNAL] Re: [satijalab/seurat] Single cell data (Issue #9561) I mean it is telling you in the error message what to look for. Your object doesn't have any scale. data layers in it, and it is asking you to run ScaleData. The tutorial is pretty clear. You import your object, you subset it based on mitochondrial I mean it is telling you in the error message what to look for. Your object doesn't have any scale.data layers in it, and it is asking you to run ScaleData. The tutorial is pretty clear. You import your object, you subset it based on mitochondrial content and counts, you run NormalizeData and then FindVariableFeatures before heading onto the next steps. I would recommend you spend a bit more time understanding what each step is actually doing, and that you aren't jumping ahead. My best guess is that you are missing earlier required steps. — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/satijalab/seurat/issues/9561*issuecomment-2555808789__;Iw!!GobTDDpD7A!LnGSdGFS8ajlcWzh6eGiAm8PhscWC78DtCvYZvLCyp5nGT6i-A5nImWG9gnaZzsJsBbnDi_0HkXdun3siRZpemmm7g$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/BN22OXAGIP2TJISPWC7XDPT2GM3NNAVCNFSM6AAAAABT5VBBAKVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDKNJVHAYDQNZYHE__;!!GobTDDpD7A!LnGSdGFS8ajlcWzh6eGiAm8PhscWC78DtCvYZvLCyp5nGT6i-A5nImWG9gnaZzsJsBbnDi_0HkXdun3siRa_eYs3Ng$>. You are receiving this because you authored the thread.Message ID: ***@***.***>

[rharao]

@samuel-marsh or anyone with appropriate permissions-- can this issue be converted to a community discussion?

[samuel-marsh]

Hi @rharao I'm inclined to keep it here for now since this does involve error message (even if it ends up being command that's missing). I do want to remind everyone to please be sensitive to differing levels of R and bioinformatics expertise as this community spans beginners to experts.

@DrSawal to ensure that this is not an issue with Seurat package could you please provide the following:

• the output of sessionInfo()
• The full code you are running (beginning with object creation)
• the output is if you just run HS

Best,
Sam