Tutorial: Associating VDJ clonotyping data with scRNA-seq in Seurat
5
gravatar for jared.andrews07
6 months ago by
jared.andrews074.1k
St. Louis, MO
jared.andrews074.1k wrote:

Single cell technologies are booming, but figuring out how to integrate multimodal data sets is certainly still a challenge, even with well-supported and constantly updated software (like Seurat). I struggled to find any info as to how to associate my Chromium 10X TCR-sequencing data with the corresponding scRNA-seq data. Seurat has a fairly good vignette on associating multimodal data, but that approach doesn't really work for V(D)J cell profiling.

The author's commented that the way to do it was by adding to the metadata of a Seurat object, but the method to do that remained unclear (partially due to the AddMetaData function not having great documentation). In reality, it's pretty easy - you can add any dataframe as Metadata to a Seurat object so long as its row names match the objects as shown with object@meta.data.

The VDJ info from 10X takes some amount of fanagling to get in a proper data frame with 1 row per cell with usable clonotype info. This R function makes it straightforward for a typical CellRanger3 workflow used for both scRNA and TCR-seq and Seurat v3:

add_clonotype <- function(tcr_location, seurat_obj){
    tcr <- read.csv(paste(tcr_folder,"filtered_contig_annotations.csv", sep=""))

    # Remove the -1 at the end of each barcode.
    # Subsets so only the first line of each barcode is kept,
    # as each entry for given barcode will have same clonotype.
    tcr$barcode <- gsub("-1", "", tcr$barcode)
    tcr <- tcr[!duplicated(tcr$barcode), ]

    # Only keep the barcode and clonotype columns. 
    # We'll get additional clonotype info from the clonotype table.
    tcr <- tcr[,c("barcode", "raw_clonotype_id")]
    names(tcr)[names(tcr) == "raw_clonotype_id"] <- "clonotype_id"

    # Clonotype-centric info.
    clono <- read.csv(paste(tcr_folder,"clonotypes.csv", sep=""))

    # Slap the AA sequences onto our original table by clonotype_id.
    tcr <- merge(tcr, clono[, c("clonotype_id", "cdr3s_aa")])

    # Reorder so barcodes are first column and set them as rownames.
    tcr <- tcr[, c(2,1,3)]
    rownames(tcr) <- tcr[,1]
    tcr[,1] <- NULL

    # Add to the Seurat object's metadata.
    clono_seurat <- AddMetaData(object=seurat_obj, metadata=tcr)
    return(clono_seurat)
    }

This saves a clonotype ID and CDR3 AA sequence, as the clonotype ID may not be usable as an identifier if samples are combined.

Hopefully this saves someone a bit of time.

ADD COMMENTlink modified 5 months ago by atakanekiz180 • written 6 months ago by jared.andrews074.1k

Since you had some confusion about AddMetaData, I just wanted to add that object@meta.data is just a data frame. If you are more comfortable with standard R data frames, you can do something like

object@meta.data$new_col <- some_values

or

object@meta.data[cell_barcodes, "new_col"] <- my_df[cell_barcodes, "some_col"]

to make sure that you are using the same cell barcodes for the labels.

Before v3, the AddMetaData function was actually relatively simple: https://github.com/satijalab/seurat/blob/65b77a9480281ef9ab1aa8816f7c781752092c18/R/interaction.R#L1120-L1137

ADD REPLYlink modified 6 months ago • written 6 months ago by igor8.9k

Thanks for the addition. Yeah, I actually went back to v2 to figure it out. I'm not quite sure why they changed the documentation to something that's (imo) more confusing, but you're dead-on that it's really simple once you realize it's just a data frame.

ADD REPLYlink written 6 months ago by jared.andrews074.1k

Thanks for this really helpful tutorial. I have now added my VDJ data to two seurat objects ("before" and "after" treatment) and then intergrated them. I now have two questions that I was wondering if you could help me with please? I would like to subset my Seurat object so that I retain the top 10 most abundant clones from "before" and "after" so that I can compare their gene expression. However, whilst I can see in the $clonotype_id that each cell has the information e.g.

head(immune.combined$clonotype_id) AAACGGGAGTGGGTTG AAAGTAGAGAAACGAG AAAGTAGAGACTGGGT AAAGTAGCAGTAAGCG "clonotype25" "clonotype6" "clonotype1" NA AAATGCCAGAGATGAG AACACGTCAGATTGCT "clonotype1" "clonotype26"

(where clonotype1 was the most abundant clonotype) I can't work out how to subset on "clonotype1", "clonotype2" etc. Please could you tell me how to do this? Also, each of the original seurat objects had its own clonotype1, clonotype2 etc. When I integrated them will they have been renamed or will clonotype1 now refer to two different cdr3s, one from "before" and one from "after"? If so, how would I go about subsetting the clonotype that I am interested in from just one condition. Many thanks in advance for your help.

ADD REPLYlink written 21 days ago by Hanna10
1

The clonotype IDs will repeat on a sample-by-sample basis, that's why I grab the sequences here, as they are directly comparable. To elaborate, clonotype 1 for one sample will not be the same as clonotype 1 from another sample.

Do you want to compare the 10 most abundant clones from "before" and the 10 most abundant from "after", or just the 10 most abundant clones overall (but compared between the two groups)?

You can see how to subset Seurat objects here (click the Seurat Object Interaction tab).

ADD REPLYlink written 21 days ago by jared.andrews074.1k
1

Thanks, for some reason I was trying to make subsetting the seurat object far more complicated than it needed to be and your link solved it for me. I wanted to subset on the top 10 from each group, so this was easy to do as the names have remained the same, but now I can see that if I do want to look at just the top 10 overall I could subset on the cdr3s_aa by the specific aa sequence. Thanks again for your help!

ADD REPLYlink written 20 days ago by Hanna10
0
gravatar for atakanekiz
5 months ago by
atakanekiz180
atakanekiz180 wrote:

@j-andrews7 thanks for this vignette. I expanded it further to work with my own data.

The detailed tutorial can be found at my Biostars post.

ADD COMMENTlink written 5 months ago by atakanekiz180
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