no plots available for ADT data when using ADT + HTO

[m3lorra]

Hi all, I hope you can help me here. I've been following your tutorials to set up my data in the following order:

mat <- as.data.frame(as.matrix(mat.so@assays$RNA@data))
mat.adt <- as.data.frame(as.matrix(mat.so@assays$ADT@data))
mat.hto <- as.data.frame(as.matrix(mat.so@assays$HTO@data))

htos <- hto.anno(hto.data = mat.hto, cov.thr = 10, assignment.thr = 80)
htos <- subset(htos,htos$percent.match > 80)

# cell ids to hashtags
sample1 <- row.names(subset(htos,htos$assignment.annotation == "HTO1"))
sample1.rna <- mat[ , which(names(mat) %in% sample1)]
my.data <- data.aggregation(samples = c("sample1.rna","sample2.rna"), 
   condition.names = c("c","t"))
mat.icellr <- make.obj(my.data)

# Then add the adt data frame
mat.icellr <- add.adt(mat.icellr, adt.data = mat.adt)

# following with qc.stat and cell.filter

# normalize RNA
mat.icellr <- norm.data(mat.icellr, norm.method = "ranked.glsf", top.rank = 500) 
# normalize ADT
mat.icellr <- norm.adt(mat.icellr)

# 2nd QC 
mat.icellr <- qc.stats(mat.icellr,which.data = "main.data")
# gene stats
mat.icellr <- gene.stats(mat.icellr, which.data = "main.data")
# genes for PCA
# merge RNA + ADT
mat.icellr <- adt.rna.merge(mat.icellr, adt.data = "main")
# run PCA and so on. 

Using this workflow I cannot plot anything using the "ADT_ab". However, when I use the ADT data alone, I'm able to do it. I checked the dataframe from both objects and they seem the same, just that when I use HTO I got less cells as I removed duplicates and negatives

Object with ADT + HTO

###################################
,--. ,-----.       ,--.,--.,------.
`--''  .--./ ,---. |  ||  ||  .--. '
,--.|  |    | .-. :|  ||  ||  '--'.'
|  |'  '--'\   --. |  ||  ||  |
`--' `-----' `----'`--'`--'`--' '--'
###################################
An object of class iCellR version: 1.6.5
Raw/original data dimentions (rows,columns): 32285,11236
Data conditions in raw data: o,y (5822,5414)
Row names: 0610005C13Rik,0610006L08Rik,0610009B22Rik ...
Columns names: y_AAACCCAAGCAGGCAT,y_AAACCCACAGCTCTGG,y_AAACCCATCGCTGTCT ...
###################################
   QC stats performed:TRUE, PCA performed:TRUE
   Clustering performed:FALSE, Number of clusters:0
   tSNE performed:FALSE, UMAP performed:TRUE, DiffMap performed:FALSE
   Main data dimensions (rows,columns): 32310,9840
   Data conditions in main data:o,y(4958,4882)
   Normalization factors:0.689367228711468,...
   Imputed data dimensions (rows,columns):0,0
############## scVDJ-seq ###########
VDJ data dimentions (rows,columns):0,0
############## CITE-seq ############
   ADT raw data  dimensions (rows,columns):25,13190
   ADT main data  dimensions (rows,columns):25,13190
   ADT columns names:AAACCCAAGCAGGCAT...
   ADT row names:ADT_B220...
############## scATAC-seq ############
   ATAC raw data  dimensions (rows,columns):0,0
   ATAC main data  dimensions (rows,columns):0,0
   ATAC columns names:...
   ATAC row names:...
############## Spatial ###########
Spatial data dimentions (rows,columns):0,0
########### iCellR object ##########

head([email protected])[1:3]
          AAACCCAAGCAGGCAT AAACCCAAGTATGATG AAACCCAAGTCGAATA
ADT_B220          0.000000          0.00000         0.000000
ADT_CD115         9.870273          0.00000         3.290091
ADT_CD11b         0.000000         16.85752       101.145095
ADT_CD11c         0.000000         90.52983         0.000000
ADT_CD127         0.000000          0.00000        77.677947
ADT_Flt3         27.195452         45.32575        27.195452

Object with ADT only

###################################
,--. ,-----.       ,--.,--.,------.
`--''  .--./ ,---. |  ||  ||  .--. '
,--.|  |    | .-. :|  ||  ||  '--'.'
|  |'  '--'\   --. |  ||  ||  |
`--' `-----' `----'`--'`--'`--' '--'
###################################
An object of class iCellR version: 1.6.5
Raw/original data dimentions (rows,columns): 32285,13190
Data conditions: no conditions/single sample
Row names: Xkr4,Gm1992,Gm19938 ...
Columns names: AAACCCAAGCAGGCAT,AAACCCAAGTATGATG,AAACCCAAGTCGAATA ...
###################################
   QC stats performed:TRUE, PCA performed:TRUE
   Clustering performed:FALSE, Number of clusters:0
   tSNE performed:FALSE, UMAP performed:TRUE, DiffMap performed:FALSE
   Main data dimensions (rows,columns): 32310,11278
   Normalization factors:0.640626466090974,...
   Imputed data dimensions (rows,columns):0,0
############## scVDJ-seq ###########
VDJ data dimentions (rows,columns):0,0
############## CITE-seq ############
   ADT raw data  dimensions (rows,columns):25,13190
   ADT main data  dimensions (rows,columns):25,13190
   ADT columns names:AAACCCAAGCAGGCAT...
   ADT row names:ADT_B220...
############## scATAC-seq ############
   ATAC raw data  dimensions (rows,columns):0,0
   ATAC main data  dimensions (rows,columns):0,0
   ATAC columns names:...
   ATAC row names:...
############## Spatial ###########
Spatial data dimentions (rows,columns):0,0
########### iCellR object ##########

head([email protected])[1:3]
          AAACCCAAGCAGGCAT AAACCCAAGTATGATG AAACCCAAGTCGAATA
ADT_B220          0.000000          0.00000         0.000000
ADT_CD115         9.870273          0.00000         3.290091
ADT_CD11b         0.000000         16.85752       101.145095
ADT_CD11c         0.000000         90.52983         0.000000
ADT_CD127         0.000000          0.00000        77.677947
ADT_Flt3         27.195452         45.32575        27.195452

If I do

A = gene.plot(my.obj, 
	gene = "ADT_Flt3",
	plot.data.type = "umap",
	interactive = F,
	cell.transparency = 0.5

Works as expected in the ADT only object, otherwise I get just a grey plot with no signal of expression. I would appreciate if you can help me to point out the reason.

Thanks!