Is it possible to compare immunarch objects with repOverlap? about immunarch

[vadimnazarov]

Hi @diyaazak

sorry for the very late response!

Can you please explain what do you mean by "immunarch objects"?

[vadimnazarov]

Ping @diyaazak about comparing single-cell repertoires in immunarch

[diyaazak]

Hi @vadimnazarov

I'm really sorry I haven't been checking this post for some time..
I'm not sure if "immunarch object" is the right term here. But what I basically do is the follows:

#load the 10x data
Bcell_immunarch = repLoad("...path/filtered_contig_annotations_TRUE.csv")

#I extract the barcodes for each cluster of cells defined by transcriptomic analysis (by Seurat for example):
Cluster_1 = c(Cluster_1_barcodes)
Cluster_2 = c(Cluster_2_barcodes)

#Cluster_1 immunarch "object"
VDJ_Cluster_1=list()
VDJ_Cluster_1$data=filter_barcodes(Bcell_immunarch$data, Cluster_1)

#Cluster_2 immunarch "object"
VDJ_Cluster_2=list()
VDJ_Cluster_2$data=filter_barcodes(Bcell_immunarch$data, Cluster_2)

#I can now use functions like geneUsage, repClonality, etc.. but only looking at each cluster separately. For example:
VDJ_Cluster_1=geneUsage(VDJ_Cluster_1$data$filtered_contig_annotations_TRUE_IGH, .norm = T)
vis(VDJ_Cluster_1)

What I would like to do for examlpe, is use functions like repOverlap to compare VDJ_Cluster_1 to VDJ_Cluster_2, or compare the gene usage between Clusters. Is there a way to do that?

Thanks a lot!

[vadimnazarov]

Hi @diyaazak

Thank you for getting back to us, our team appreciates that!

Currently you need to create a metadata table by hand and put your data in a common immunarch format as a list of two elements - data and meta. In your case:

1. Load the data
2. Filter by barcodes
3. Create new list with your data and metadata:

VDJ_Cluster_1 <- filter_barcodes(Bcell_immunarch$data[[1]], Cluster_1)
VDJ_Cluster_2 <- filter_barcodes(Bcell_immunarch$data[[1]], Cluster_2)
im_clusters <- list(data = list(VDJ_Cluster_1, VDJ_Cluster_2))
metadata <- data.frame(Sample = c("VDJ_Cluster_1", "VDJ_Cluster_2"), stringsAsFactors=F) # add any additional metadata here
im_clusters$meta <- metadata
repOverlap(im_clusters$data)

You can wrap it in a simple function to work with any number of clusters.

Full single-cell support (seamless stratification to patients and clusters by barcodes, paired chain analysis, etc.) is our next major milestone, so your feedback on your data analysis routines and goals is much appreciated!

[diyaazak]

Thanks a lot for the quick feedback!
I tried it but when I run repOverlap it doesn't seem to work:

rO=repOverlap(im_clusters$data)
rO
[1] 1
attr(,"class")
[1] "numeric" "immunr_ov_matrix"
vis(rO)

This is how my data and metadata list looks like:

head(im_clusters)
$data
$data[[1]]

A tibble: 616 x 20

Clones Proportion CDR3.nt CDR3.aa V.name D.name J.name V.end D.start D.end J.start VJ.ins VD.ins DJ.ins Sequence chain

1 2 0.00324 TGTGCA… CARRGI… IGHV1… None IGHJ3 NA NA NA NA NA NA NA TGTGCAA… IGH
2 1 0.00162 TGTGCA… CARWGE… IGHV1… None IGHJ2 NA NA NA NA NA NA NA TGTGCAA… IGH
3 2 0.00324 TGTGCA… CARSQG… IGHV1… None IGHJ1 NA NA NA NA NA NA NA TGTGCAA… IGH

… with 606 more rows, and 4 more variables: Barcode , RawClonotypeID , RawConsensusID , ContigID

$data[[2]]

A tibble: 522 x 20

Clones Proportion CDR3.nt CDR3.aa V.name D.name J.name V.end D.start D.end J.start VJ.ins VD.ins DJ.ins Sequence chain

1 2 0.00379 TGTGCA… CARHAY… IGHV1… None IGHJ4 NA NA NA NA NA NA NA TGTGCAA… IGH
2 2 0.00379 TGTGCA… CARRPW… IGHV1… None IGHJ2 NA NA NA NA NA NA NA TGTGCAA… IGH
3 2 0.00379 TGTGCA… CARDRP… IGHV5… IGHD1… IGHJ2 NA NA NA NA NA NA NA TGTGCAA… IGH

… with 512 more rows, and 4 more variables: Barcode , RawClonotypeID , RawConsensusID , ContigID

$meta
Sample
1 VDJ_Cluster_1
2 VDJ_Cluster_2

Also, geneUsage doesn't seem to work:

IgH=geneUsage(im_clusters$data, .norm = T)
Error: x must be a vector, not a tbl_df/tbl/data.frame/immunr_gene_usage object.

I wonder what I might be doing wrong.
Thanks for your support!

[vadimnazarov]

Hi @diyaazak

I see, thank you. I have questions regarding the barcode preparations.
How do you get barcodes for specific clusters from Seurat?
If you have different patients, how do you store the data about barcode/patient pairs?

Regarding the analysis. Can you please name your clusters:

names(im_clusters$data) <- c("VDJ_Cluster_1", "VDJ_Cluster_2")

And try again? Immunarch requires named samples.

[vadimnazarov]

Hi @diyaazak

We made a major improvements to the package, and now you can easily split datasets by clusters and barcodes. Here is more info: https://immunarch.com/articles/web_only/v21_singlecell.html

Please let me know if it's what you seek, if it's work or if you have any other questions. Your input is much appreciated!

[diyaazak]

Hi @vadimnazarov
Thanks a lot for the follow-up and I am sorry for not writing you earlier. This worked finally!
I tried most of the functions and they work fine. I think only plotting geneUsage wouldn't make sense now that the chains are paired (you get too many).
I didn't get how the data was structured in the provided "scdata" example though. I prepared my data as follows in the end:

VDJ <- repLoad(.path = "...path/filtered_contig_annotations_TRUE.csv", .mode = "paired")
Cluster1 <- c("Cluster1_barcodes") #I extract the barcodes manually from Seurat metadata
Cluster2 <- c("Cluster2_barcodes")
VDJ_Cluster_1 <- select_barcodes(VDJ$data[[1]], Cluster1)
VDJ_Cluster_2 <- select_barcodes(VDJ$data[[1]], Cluster2)

im_clusters <- list(data = list(VDJ_Cluster_1, VDJ_Cluster_2))
names(im_clusters$data) <- c("VDJ_Cluster_1", "VDJ_Cluster_2")
metadata <- data.frame(Sample = c("VDJ_Cluster_1", "VDJ_Cluster_2"), stringsAsFactors=F) 
im_clusters$meta <- metadata

Is this the way you would perform it or is there a more straightforward way?

I tried running select_clusters as follows but it doesn't seem to work:

scdata_pat <- select_clusters(VDJ, my_seurat_object$seurat_clusters )

Thanks!

[vadimnazarov]

Hi @diyaazak

thank you! Can you print my_seurat_object$seurat_clusters into the Console and send me the output, please?

Can you also send me the error message after scdata_pat <- select_clusters(VDJ, my_seurat_object$seurat_clusters ), please?

[diyaazak]

head(my_seurat_object$seurat_clusters)
AAACCTGAGAAGATTC AAACCTGAGAGACGAA AAACCTGAGGGCTCTC AAACCTGCAAACAACA
2 0 1 3
AAACCTGCACCCATTC AAACCTGCACCGAAAG
1 0
Levels: 0 1 2 3 4 5 6 7 8 9 10 11

Now that I look at it, it could be because the barcodes in the filtered_contig_annotations.csv file have a -1 at the end while the barcodes from a Seurat object lack that. I only started learning R recently so I was just doing this manually in an excel sheet before using select_barcodes

I don't get an error when I run scdata_pat <- select_clusters(VDJ, my_seurat_object$seurat_clusters ). I just get a list with empty data..the metadata is there though:

scdata_cl
$data
$data$filtered_contig_annotations_0
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_1
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_2
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_3
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_4
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_5
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_6
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_7
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_8
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_9
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_10
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$data$filtered_contig_annotations_11
[1] CDR3.nt CDR3.aa V.name D.name
[5] J.name V.end D.start D.end
[9] J.start VJ.ins VD.ins DJ.ins
[13] Sequence chain raw_clonotype_id ContigID
[17] Clones Barcode Proportion
<0 rows> (or 0-length row.names)

$meta

A tibble: 12 x 3

Sample Cluster.source Cluster

1 filtered_contig_annotations_0 filtered_contig_annotations 0
2 filtered_contig_annotations_1 filtered_contig_annotations 1
3 filtered_contig_annotations_2 filtered_contig_annotations 2
4 filtered_contig_annotations_3 filtered_contig_annotations 3
5 filtered_contig_annotations_4 filtered_contig_annotations 4
6 filtered_contig_annotations_5 filtered_contig_annotations 5
7 filtered_contig_annotations_6 filtered_contig_annotations 6
8 filtered_contig_annotations_7 filtered_contig_annotations 7
9 filtered_contig_annotations_8 filtered_contig_annotations 8
10 filtered_contig_annotations_9 filtered_contig_annotations 9
11 filtered_contig_annotations_10 filtered_contig_annotations 10
12 filtered_contig_annotations_11 filtered_contig_annotations 11

Thanks!

[vadimnazarov]

Hi @diyaazak

It definitely can be a case since barcode matching is straightforward and not "smart" to check for "-1" etc. You can easily add "-1" to your barcodes by (note that I don't change the Seurat object here):

barcode_vec <- my_seurat_object$seurat_clusters
names(barcode_vec) <- paste0(names(barcode_vec), "-1")
scdata_pat <- select_clusters(VDJ, barcode_vec)

Thank you for pointing us to that issue, we will fix it by removing "-1" in the next release for the convenience

[vadimnazarov]

Hi @diyaazak

Did it work?

[diyaazak]

Hi @vadimnazarov
Yes it did! Thanks for the great help.

[Alexander230]

Hello,
My name is Aleksandr Popov, I am a developer of the Immunarch package. Thank you for using our software!

There was no activity on this issue for a while, so we plan to close it in 1 month. You are welcome to comment and reopen the issue if there are still unresolved questions.