Pseudotime trajectory analysis not working, throwing error.

[ruchikabhat]

Hi,

I am using pseudotime analysis for the first time for mouse scRNA data and have encountered the following error:

Here is my code:

library(Seurat)
library(SeuratDisk)
library(SeuratData)
library(ggplot2)
library(monocle)
library(slingshot)
library(BUSpaRse)
library(RColorBrewer)
set.seed(1)
n <- 30
qual_col_pals = brewer.pal.info[brewer.pal.info$category == 'qual',]
col_vector = unlist(mapply(brewer.pal, qual_col_pals$maxcolors, rownames(qual_col_pals)))
a<-DiscretePalette(n, palette = NULL)

######load the seurat object first ######
setwd ("D:/HPC_DATA_MOUSE_NEW COVID_April2022/Rrun")
mouse<-readRDS("mouseAnnotated_16thMarch2023.rds")     ##NEWEST ##

Idents(mouse)<-'seurat_clusters'
FRC<-subset(mouse,idents= c('6'), invert=TRUE)
Idents(FRC)<-'Condition'
Ad<-subset(FRC,idents= c('Adult'), invert=FALSE)
Od<-subset(FRC,idents= c('Old'), invert=FALSE)
Idents(Ad)<-'Ref_celltypes'
Lec_aad<-subset(Ad,idents= c('LECs'), invert=FALSE)
Idents(Lec_aad)<-'Clusters'
Lec_ad<-subset(Lec_aad,idents= c('Cd45+Bcells','Cd45+Tcells','NerveCells'), invert=TRUE)

View(Lec_ad@meta.data)
Idents(Lec_ad)<-'Clusters'
data <- as(as.matrix(Lec_ad@assays$RNA@data), 'sparseMatrix')
pd <- new('AnnotatedDataFrame', data = Lec_ad@meta.data)
fData <- data.frame(gene_short_name = row.names(data), row.names = row.names(data))# save list of gene names
fd <- new('AnnotatedDataFrame', data = fData)

############ NEWCELLDATASET ###########
FibroMono <- newCellDataSet(data,
                            phenoData  = pd,
                            featureData = fd,expressionFamily = negbinomial.size())

####ESTIMATE DISPERSIONS AND SIZE FACTORS############
FibroMono <- estimateSizeFactors(FibroMono)
FibroMono <- estimateDispersions(FibroMono)

#######FILTER LOW QUALITY GENES/CELLS##########
FibroMono <- detectGenes(FibroMono, min_expr = 0.1)
print(head(fData(FibroMono)))

expressed_genes <- row.names(subset(fData(FibroMono),
                                    num_cells_expressed >= 10))

expressed_genes
######DEG TEST##########
diff_test_res <- differentialGeneTest(FibroMono[expressed_genes,],
                                      fullModelFormulaStr = "~Clusters")
ordering_genes <- row.names (subset(diff_test_res, qval < 0.01))
ordering_genes

################## Run ordering algorithm #############
FibroMono <- setOrderingFilter(FibroMono, ordering_genes)
print(dim(exprs(FibroMono)))

## reduce dimension - do not normalize or include pseudo count. Use monocle scaling
FibroMono <- reduceDimension(FibroMono,norm_method="none", 
                             reduction_method="DDRTree",
                             max_components=2,
                             scaling=TRUE,
                             verbose=TRUE,
                             pseudo_expr=0, auto_param_selection=F)

# First decide what you want to color your cells by
print(head(pData(FibroMono)))

## order cells change colors and theta to match your plot
FibroMono <- orderCells(FibroMono)

## Plot Pseudotime trajectories ##
plot_ordering_genes(FibroMono)
plot_cell_trajectory(FibroMono, 
                     color_by = "Clusters",
                     theta = -15,
                     show_branch_points = TRUE,show_backbone = TRUE,
                     backbone_color = "black",
                     show_tree = TRUE,use_color_gradient = FALSE,
                     cell_size = 1.5, values=a) + scale_color_manual(values = a, name = "Only FRCs Pseudotime")+ theme(legend.position = "top")

FibroMono <- orderCells(FibroMono)

Warning messages: 1: In graph.dfs(dp_mst, root = root_cell, neimode = "all", unreachable = FALSE, : Argument neimode' is deprecated; use mode' instead 2: In graph.dfs(dp_mst, root = root_cell, neimode = "all", unreachable = FALSE, : Argument neimode' is deprecated; use mode' instead

plot_cell_trajectory(FibroMono,

                 color_by = "Clusters",
                 theta = -15,
                 show_branch_points = TRUE,show_backbone = TRUE,
                 backbone_color = "black",
                 show_tree = TRUE,use_color_gradient = FALSE,
                 cell_size = 1.5, values=a) + scale_color_manual(values = a, name = "Only FRCs Pseudotime")+ theme(legend.position = "top")

Error in .standalone_types_check_dot_call(ffi_standalone_check_number_1.0.7, : object 'ffi_standalone_check_number_1.0.7' not found

Please help in solving the problem. How to know/plot the pseudotime of these celltypes? Any help would be highly appreciated.

Thank you in advance.

[HangboZhu]

same issues

[HangboZhu]

I update the packages which name is "rlang" to '1.1.0' ,then the problem was solved.

[ruchikabhat]

@Humboler .. Thanks a lot for your kind help! It worked.