Error with 'replacement has 1378 rows, data has 1345'

[biansr]

Hello, I've been recently tried to use singleR but it failed after the step creating a singleR object. My seurat object contains 14793 genes and 1378 cells. I use:

singler = CreateSinglerObject(syno@data, annot = NULL, syno, min.genes = 500, technology = "10X", species = "Human", citation = "", ref.list = list(), normalize.gene.length = F, variable.genes = "de", fine.tune = T, do.signatures = F, clusters = NULL, do.main.types = T, reduce.file.size = T, numCores = SingleR.numCores) singler$seurat <- syno singler$meta.data$xy <- ra.control@dr$tsne@cell.embeddings singler$meta.data$clusters <- ra.control@ident

to create the singleR object, and:

SingleR.DrawHeatmap(singler$singler[[1]]$SingleR.single.main,top.n=Inf, clusters = singler$meta.data$clusters) out = SingleR.PlotTsne(singler$singler[[1]]$SingleR.single.main, singler$meta.data$xy,do.label=FALSE, do.letters =T,labels=singler$singler[[1]]$SingleR.single.main$labels, dot.size = 2, font.size = 12) out$p

for analysis. However, it returns with an error saying 'replacement has 1378 rows, data has 1345'. Do you know what might cause the data read 33 less rows? Thank you very much.

Best, Shurui.

[dviraran]

Hi, Its probably because of using a different threshold for min.genes. When using an already available Seurat object you don't want any filtering of the genes. Just use mig.genes = 0.

I'll change it in the example as well.

Best, Dvir

[biansr]

Hi, Its probably because of using a different threshold for min.genes. When using an already available Seurat object you don't want any filtering of the genes. Just use mig.genes = 0.

I'll change it in the example as well.

Best, Dvir

Thank you very much! It solves the issue. Now I have another issue. When I tried:

out = SingleR.PlotTsne(singler$singler[[1]]$SingleR.single.main, singler$meta.data$xy,do.label=FALSE, do.letters =T,labels=singler$singler[[1]]$SingleR.single.main$labels, dot.size = 2, font.size = 12) out$p

to plot tSNE plot, it returns an error of 'Error in FUN(X[[i]], ...) : object 'x' not found'. Do you have any idea why that would happen?

[dviraran]

Great.

Its hard to guess... looking back at your code singler$meta.data$xy seems to be taken from a different object than the seurat object. Is it set properly?

Can you print out the traceback() output?

[biansr]

What do you mean by saying "taken from a different object than the seurat object"? Actually I'm a bit confused too, because singler$meta.data seems to only have two components: project.name, and orig.ident. singler$meta.data$xy returns NULL. Maybe that's something that I need to change from the sample code? However both using project.name and orig.ident return errors.

traceback()

11: FUN(X[[i]], ...) 10: lapply(aesthetics[new_aesthetics], rlang::eval_tidy, data = data) 9: scales_add_defaults(plot$scales, data, aesthetics, plot$plot_env) 8: f(..., self = self) 7: l$compute_aesthetics(d, plot) 6: f(l = layers[[i]], d = data[[i]]) 5: by_layer(function(l, d) l$compute_aesthetics(d, plot)) 4: ggplot_build.ggplot(x) 3: ggplot_build(x) 2: print.ggplot(x) 1: function (x, ...) UseMethod("print")(x)

[dviraran]

I guessed right... :)

You have the seurat object called syno, but to copy those you are a different object (ra.control).

singler$meta.data$xy <- ra.control@dr$tsne@cell.embeddings singler$meta.data$clusters <- ra.control@ident

those lines just copy the tSNE coordinates and the cluster identities from Seurat. You need to figure out that you are doing it correctly...

[biansr]

I guessed right... :)

You have the seurat object called syno, but to copy those you are a different object (ra.control).

singler$meta.data$xy <- ra.control@dr$tsne@cell.embeddings singler$meta.data$clusters <- ra.control@ident

those lines just copy the tSNE coordinates and the cluster identities from Seurat. You need to figure out that you are doing it correctly...

What a terrible mistake I've made. Thank you very much!