edroaldo / cellrouter Goto Github PK

Hello, I seem to be getting the same error as "processtrajectories error #9" however I am following the Paul example using that data and copy pasting the the exact script from the tutorial. All seemed to be going quite well until this step. Please advise, thanks so much.

Here is the command and output I am seeing:

cellrouter <- processTrajectories(cellrouter, rownames(cellrouter@ndata), path.rank=ranks[3], num.cells = 3, neighs = 3,column.ann = 'population', column.color = 'colors')
[1] "parsing trajectory information"
[1] 3
Error in igraph::induced.subgraph(graph = g, vids = unlist(igraph::neighborhood(graph = g, :
Not a graph object

here is some other info I noticed you requested in the other thread:

user@server:~$ ls results
custom_1.pdf pathsCells_FlowNetwork_paths.ser
heatmap_all_genes_CellRouter_clusters.png PCA_1.pdf
heatmap_top_10_genes_CellRouter_clusters.png PCA_2.pdf
heatmap_top_10_genes_original_clusters.png tSNE_1.pdf
heatmap_top_10_genes_sorted_populations.png tSNE_2.pdf
knn_tsne_CellRouter_clusters.pdf tSNE_graphClustering_clusters.pdf
paths tSNE_original_clusters.pdf

user@server:~$ ls results/paths
6.1 6.2 6.3 6.4 6.5 6.7 6.8 6.9 cell_edge_weighted_network.txt

user@server:~$ java -version
openjdk version "11.0.7" 2020-04-14
OpenJDK Runtime Environment (build 11.0.7+10-post-Ubuntu-2ubuntu218.04)
OpenJDK 64-Bit Server VM (build 11.0.7+10-post-Ubuntu-2ubuntu218.04, mixed mode, sharing)

user@server:~$ ls results/paths/6.1
CellRouter.sh Cells_FlowNetwork_scripts.R
Cells_FlowNetwork_all_paths_subnet_exportGML.R Cells_FlowNetwork_scripts.Rout
Cells_FlowNetwork_all_paths_subnet.txt cell_sinks.txt
Cells_FlowNetwork_all_paths_totalFlow.txt cell_sources.txt
Cells_FlowNetwork_all_paths.txt

then I found in the Cells_FlowNetwork_scripts.Rout file that it was stalling when attempting to load igraph:

library(igraph);
Error in library(igraph) : there is no package called ‘igraph’
Execution halted

this is because I have separate Rlibs directories on this server and so it needed to load my libPaths() first. I added that line to the CellRouter_Class.R script:

setMethod("processTrajectories",
signature="CellRouter",
definition=function(object, genes, path.rank, num.cells, neighs, column.ann, column.color){
.libPaths(c("wsRlibs361", .libPaths()))
library(igraph)
[email protected] <- genes

and it fixed that problem? but introduced what seems to be a worse one at this earlier step:

cellrouter <- findPaths(cellrouter, column='population', libdir, paste(getwd(), 'results/paths', sep='/'), method="graph")
-------------------Transition: 6.1 -----------------------

1. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter
   -------------------Transition: 6.2 -----------------------
2. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter
   -------------------Transition: 6.3 -----------------------
3. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter
   -------------------Transition: 6.4 -----------------------
4. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter
   -------------------Transition: 6.5 -----------------------
5. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter
   -------------------Transition: 6.7 -----------------------
6. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter
   -------------------Transition: 6.8 -----------------------
7. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter
   -------------------Transition: 6.9 -----------------------
8. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter
   -------------------Transition: 6.10 -----------------------
9. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   Caused by: java.lang.ClassNotFoundException: cellrouter.CellRouter

I thought it was a server related problem however I also tried the same wokflow on my macbook and at the same step got this

cellrouter <- findPaths(cellrouter, column='population', libdir, paste(getwd(), 'results/paths', sep='/'), method="graph")
-------------------Transition: 6.1 -----------------------

1. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   -------------------Transition: 6.2 -----------------------
2. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter

Now I am totally lost. Please let me know if there is anything else that would help get to the bottom of this. Thanks again.
W

Dear Edroaldo,
Thanks for designing the cellrouter program. Recently, I'm try to do trajectory using it.
My current data is from Seurat, but I met this error when I want to create a CellRouter object.
My code is here:

samples <- get(load('Paul_Cell_samples.R'))
cellrouter <- CellRouter(path='Paul_Cell_data.R', min.cells=3, min.genes=0)
[1] "Initializing CellRouter object"
data <- as.data.frame(as.matrix([email protected]))
cellrouter <- CellRouter(rawdata=as.data.frame(data), min.cells=0, min.genes=0)
[1] "Initializing CellRouter object"
cellrouter <- addInfo(cellrouter, metadata = [email protected], colname = 'merged_20_Aug', metadata.column='res.0.8')
Error in rep(colors, times = replicate_row) : invalid 'times' argument

Any help will be appreciated! Thanks!
Best,
Kun

Hi,

Thanks again for the great work. :)
I'm testing cellrouter with our own data (Two conditions at day 3). There are ~20 cell sub-populations identified. K = 12 which was defined by findK function. I did try other K values as well.

There are relatively few clusters identified by SC3, which seems to be close to our expectation. So I'm wondering if cellrouter tends to give many sub-populations, even though the input data is collected from two conditions at single timepoint.

Looking forward to your response.

Hi,

I've been trying to run your example code but I get the following error when I get to running "buildGRN":
Error in graph.data.frame(tmpAns, directed = directed, v = verticies) : could not find function "graph.data.frame"
I had no trouble installing any dependencies either, so I don't understand where the problem could be, any insight would be appreciated.
Session info:
R version 4.0.3 (2020-10-10)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 18.04.6 LTS, RStudio 1.3.959

Hello,
Thank you for your previous answer.
While I am able to find the subpopulations with the data set you use, I am unable to do the same with my own data set (I tried with different k number). I obtain the following message.

cellrouter <- findsubpopulations (cellrouter, 5, 'jaccard')
[1] "building k-nearest neighbors graph"
[1] "discoverying subpopulation structure"

Error in simple_vs_index(x, ii, na_ok) : Unknown vertex selected

I am not able to understand the error message, I tried to find if the presence of na was a problem with the function

which(is.na(mydata), arr.ind=TRUE)

But it seems that my data set doesn't contain na
Thank you

Hi,
I'm following the tutorials and issues in github to make trajectory using Seurat object. I got this error from processTrajectories.

filename <- "results/paths/cell_edge_weighted_network.txt"
write.table(cellrouter@graph$edges, file=filename, sep='\t', row.names=FALSE, col.names = FALSE, quote=FALSE)
#Select cluster 11
sources <- c('11')
targets <- setdiff(as.vector(cellrouter@sampTab$seurat_clusters), sources)
methods <- c("euclidean", "maximum", "manhattan","canberra","binary", 'graph') #graph for distances in KNN
##Identify trajectories
cellrouter <- findPaths(cellrouter, column='seurat_clusters', libdir, paste(getwd(), 'results/paths', sep='/'), method="graph")
-------------------Transition: 11.0 -----------------------
-------------------Transition: 11.2 -----------------------
-------------------Transition: 11.3 -----------------------
-------------------Transition: 11.4 -----------------------
-------------------Transition: 11.5 -----------------------
-------------------Transition: 11.6 -----------------------
cellrouter <- processTrajectories(cellrouter, rownames(cellrouter@ndata), path.rank=ranks[3], num.cells = 3, neighs = 3, column.ann = 'seurat_clusters', column.color = 'seurat_clusters_colors')
[1] "parsing trajectory information"
Error in file(file, "rt") : cannot open the connection
In addition: Warning message:
In file(file, "rt") :

Is there a way to overlay a TSNE with the cellrouter trajectory calculated for those cells?

Hi,
When running the regulatornetwork function,Error: ggplot2 doesn't know how to deal with data of class igraph was reported.Here are the cmds I use:
> regulators=c("GATA1","GATA2")
> regulatornetwork(x, regulators, 5, 2, 5, 9, 'results/regulator_networks.pdf')
ggplot2 was upgraded to newest version 2.2.1 but the error still remained and was generated by g <- fortify(rgrn) i think. any suggestions to fix this?

Thanks a lot lot.

Hi,
sorry that i still have several questions to bother you.=.=
1.in the part of regulators network,labels of some genes are printed as picture below shows while other equal-sized ,equal-colored don't.what's meaning when a gene is labeled?

2.i'm a little confused about the definition of derivtive shown in heatmap of negtive and positive control.what's the difference and relationship with expression?

3. your article mentioned that In the neutrophil trajectory, Mxd1 is upregulated in later stages of differentiation compared to Cebpe (Fig. 2c,d). According to GRN analyses, Mxd1 is predicted to regulate Mmp8, Mmp9, Retnlg, and Cd52 expression, all of which are known neutrophil markers (Fig. 2e, f).How to infer the predictive relationship of Mxd1 from the GRN results?

Thanks for your patience and your great work sincerely.

Hi,
I work through most part of the analysis pipeline but the results of GRN scores obtained via grnscores() in specified paths SP_10.SP_8 as tSNE and barplot shows.the grn scores have different levels compared to yours presented in several exemplary analysis,that's level of 0.0* versus 0.* .Sometimes,the GRNscores plot has one or two genes with low scores in both directions.I don't know if there's some wrong configuration in my pipeline.If my source and endpoints are correct and reliable,low scores level are possible for some datasets and it's available?

another questions asking for your guide :)
known knowledge this project based on are less subpopulations and increasing number of cells along differentiation process.So,k=120 was set according to my understanding in function buildGRN() and creatKNN() to obtain relatively less subpopulations as tSNE plot shows.k value is different a lot from yours.It might be a cause of low GRN scores?And if a path didn't go through every subpopulation in non-branched cells differentiation,gene expression dynamics with this path can represent entire process of changes?

Thank a lot !

Jm Zhong

Hi,

Thanks for the great cellrouter. :)
I'm testing mouse bone marrow sc RNA-seq example code from this https://github.com/edroaldo/cellrouter/blob/master/stemid/StemID_BM_CellRouter.md. Everything works fine on my laptop. There is an error when I put them on the workstation (Linux).

Code:

processtrajectories(cellrouter, genes2use, path.rank=ranks[3], 
                                  num.cells = 3, neighs = 1)

Error:

 Error in igraph::induced.subgraph(graph = g, vids = unlist(igraph::neighborhood(graph = g,  : 
  Not a graph object

I found that the main issues come from 'cellrounter@networks = NULL', meaning no Cells_FlowNetwork_all_paths_subnet.gml files generated from CellRounter.Jar.

I checked the java version from my laptop and computer.
laptop:
java version "1.8.0_161"
Java(TM) SE Runtime Environment (build 1.8.0_161-b12)
Java HotSpot(TM) 64-Bit Server VM (build 25.161-b12, mixed mode)

computer:
openjdk version "1.8.0_131"
OpenJDK Runtime Environment (build 1.8.0_131-8u131-b11-2ubuntu1.16.04.3-b11)
OpenJDK 64-Bit Server VM (build 25.131-b11, mixed mode)

Look forward to your response. Thank you.

Hi

I am using Paul data as a toy but I am getting error :(

> plotReducedDimension(cellrouter, reduction.type = 'tsne', annotation="population", annotation.color = 'colors',showlabels = TRUE, width = 4.5, height = 3.5, filename='results/tSNE_graphClustering_clusters.pdf')
 Error in `$<-.data.frame`(`*tmp*`, "group", value = integer(0)) : 
  replacement has 0 rows, data has 2699 
> plotReducedDimension(cellrouter, reduction.type = 'tsne', annotation="celltype", annotation.color = 'celltype',showlabels = TRUE, width = 4.5, height = 3.5, filename='results/tSNE_graphClustering_clusters.pdf')
 Error in grDevices::col2rgb(colour, TRUE) : invalid color name 'CMP' 
> plotReducedDimension(cellrouter, reduction.type = 'pca', annotation="celltype", annotation.color = 'celltype',showlabels = TRUE, width = 4.5, height = 3.5, filename='results/tSNE_graphClustering_clusters.pdf')
 Error in grDevices::col2rgb(colour, TRUE) : invalid color name 'CMP' 

Can you help please?

Hello,
I ran findpaths funtion with imputed 10x matrix according to the example with no error before this step.But the 2 generated files in results\paths\SP_46.SP_* are nearly empty.CellRouter.sh file are complete but executed unsuccessfully with "running command './CellRouter.sh' had status 127" in Rstudio with R 3.4.3 version.Any idea to this issue?

Thanks in advance,
Jianmei Zhong

Sorry,

This is not a problem with your nice software rather my own problem.

I noticed by plotDRExpression function we can plot a list of genes very clean because you are using a sort of centring I guess (-2,2).

You please imagine that, I have made 3 clusters of cells (clusters 1, 2 and 3) . I expect if I plot a list of marker genes across these three clusters, only cluster 1 should be lighted up because these marker genes supposed to be specific to cluster 1. Is there any way to use your plotDRExpression function for a list of genes but not each of them individually rather something like mean of expression of them or whatever by which I could have such a nice featureplot showing the expression of these genes?

I tried seurat like below code but my way of centring another clusters also being lighted up that I don't want

mat=seurat_object@data[rownames(seurat_object@data)%in%rownames(gene_list),]   
 
#I have converted expression matrix to a binary matrix by 2 as a threshold (arbitrarily)

mat[mat < 2] <- 0

mat[mat > 2] <- 1

 

 
exp=colSums(mat)

exp=as.matrix(exp)

colnames(exp)="value"

exp=as.data.frame(exp)

cc <- AddMetaData(object = surat_object, metadata = exp)

cc <- SetAllIdent(object = cc, id = "value")

TSNEPlot(object = cc, do.return= TRUE)

ggplot(as.data.frame([email protected]), aes(x = [email protected]$CELL, y = [email protected]$res.0.7, colour [email protected]$value)) + 
       geom_point(size = 9) +
      scale_colour_gradient(low = "yellow", high = "blue")

Thanks a lot for any help

When I run:

genelist <- c('Hspg2', 'Kdr', 'Plvap', 'Gja4')
plottrajectories(cellrouter, '8.9', genelist, rescale = TRUE, columns=1, width=5, height=2, filename='results/dynamics_curve.pdf')

the plottrajectories function runs just fine.

When I try to run plotPathHeatmap with the same genelist:

genelist <- c('Hspg2', 'Kdr', 'Plvap', 'Gja4')
plotPathHeatmap(cellrouter, '8.9', genelist, 2, 4, 2, dir = 'results/')

I get the error:

Error in `.rowNamesDF<-`(x, value = value) : invalid 'row.names' length 

How can I make a heatmap with the desired genes?

Very nice work! However I am trying to work with your package and it goes smooth until the step of "processtrajectories" in which I am getting this error:

cellrouter <- processtrajectories(cellrouter, genes2use, path.rank=ranks[3],num.cells = 3, neighs = 1)
[1] "parsing trajectory information"
Error in complete.cases(paths) :
no input has determined the number of cases

Any idea ?

Best,
natlasy

Hi,

I was trying the demon code under Linux with Oracle java, but encounter the R-running issue below:
cellrouter <- findPaths(cellrouter, column='label', libdir, paste(ge
-------------------Transition: 12.9 -----------------------

1. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter
   -------------------Transition: 12.3 -----------------------
2. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter

I tried both R/3.6.3 and R/4.0.3; both met the same error
$ java -version
java version "1.8.0_92"
Java(TM) SE Runtime Environment (build 1.8.0_92-b14)
Java HotSpot(TM) 64-Bit Server VM (build 25.92-b14, mixed mode)

Thank you for helping
Holly

HI,

I'm trying to find paths for my custom_clusters, that are added to the cellrouter@sampTab:

cellrouter <- findPaths(cellrouter, column='custom_clusters', libdir, paste(getwd(), 'results/paths', sep='/'), method="graph")

and it generates an error:

Error in order(tmp2$value, decreasing = TRUE) :
argument 1 is not a vector

How could I resolve the issue?

Thank you,
Araks

Hi,

I am trying to use cell router for trajectory analysis with Seurat object. Here are the commands and the errors that I am getting.

intestinal_organoids_corrected_metadata <- [email protected]

intestinal_organoids_corrected_data <- as.data.frame(as.matrix(intestinal_organoids_corrected@data))
intestinal_organoids_corrected_cellrouter <- CellRouter(rawdata=as.data.frame(intestinal_organoids_corrected_data), min.cells=0,min.genes=0)
intestinal_organoids_corrected_cellrouter <- addInfo(intestinal_organoids_corrected_cellrouter, metadata = intestinal_organoids_corrected_metadata, colname = 'res.0.9', metadata.column='res.0.9')
intestinal_organoids_corrected_cellrouter <- addInfo(intestinal_organoids_corrected_cellrouter, metadata = intestinal_organoids_corrected_metadata, colname = 'class', metadata.column='class')

plotReducedDimension(intestinal_organoids_corrected_cellrouter, reduction.type = 'tsne', dims.use =
c(1,2), annotation = "res.0.9", annotation.color ='res.0.9_color', showlabels = TRUE, 4.5, 3.5,
filename='intestinal_organoids_corrected_tsne_seurat_clusters_cellrouter.pdf')

Error in names(x) <- value :
'names' attribute [2] must be the same length as the vector [0]

#for trajectory analysis:
intestinal_organoids_corrected_cellrouter <- buildKNN(intestinal_organoids_corrected_cellrouter, k = 10, column.ann = 'res.0.9',
num.pcs = 20, sim.type = 'jaccard')

Error in dist(matrix) :
Can only handle data frames, vectors, matrices, and lists!

plotKNN(cellrouter, reduction.type = 'tsne', column.ann = 'res.0.9',
column.color = 'res.0.9_color', width = 5, height = 4,
filename='results/knn_tsne_original_clusters.pdf')

Appreciate your help.

Thanks,

Bibaswan

Hi,edroaldo,
Thanks sincerely your previous helpful solutions again.Here I ask for your help again but it's in the part of running davidclustering function. :)
the error reported like this:

cellrouter=davidclustering(cellrouter,paths,"./","./",email=NULL,ids)
Error in fea_david(names(x), annotations = c("GOTERM_BP_ALL", "KEGG_PATHWAY", : Query url too long.

ids is a data frame generated by myself with two colmuns 'external_gene_name' and 'ensembl_gene_id' which contain all 432 clustering genes in cellrouter@clusters$paths$clustering.

sessionInfo()
R version 3.4.3 (2017-11-30)
Platform: x86_64-conda_cos6-linux-gnu (64-bit)
Running under: Red Hat Enterprise Linux Server release 6.4 (Santiago)
Matrix products: default
BLAS/LAPACK: /root/miniconda2/lib/R/lib/libRblas.so
locale:
[1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
[5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=en_US.UTF-8
[9] LC_ADDRESS=en_US.UTF-8 LC_TELEPHONE=en_US.UTF-8
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=en_US.UTF-8
attached base packages:
[1] grid stats4 parallel stats graphics grDevices utils
[8] datasets methods base
other attached packages:
[1] bitops_1.0-6 genefilter_1.60.0 gplots_3.0.1
[4] mclust_5.4 plyr_1.8.4 igraph_1.2.1
[7] tsne_0.1-3 plotrix_3.7 ReactomePA_1.22.0
[10] clusterProfiler_3.6.0 DOSE_3.4.0 pheatmap_1.0.8
[13] reshape2_1.4.3 reshape_0.8.7 RDAVIDWebService_1.16.0
[16] ggplot2_2.2.1 GOstats_2.44.0 Category_2.44.0
[19] Matrix_1.2-12 AnnotationDbi_1.40.0 IRanges_2.12.0
[22] S4Vectors_0.16.0 Biobase_2.38.0 graph_1.56.0
[25] BiocGenerics_0.24.0 rJava_0.9-9 FGNet_3.12.0
[28] RCurl_1.95-4.10
loaded via a namespace (and not attached):
[1] httr_1.3.1 tidyr_0.8.0 bit64_0.9-7
[4] splines_3.4.3 R.utils_2.6.0 gtools_3.5.0
[7] DO.db_2.9 rvcheck_0.0.9 RBGL_1.54.0
[10] blob_1.1.1 pillar_1.2.1 RSQLite_2.0
[13] backports_1.1.2 lattice_0.20-35 glue_1.2.0
[16] digest_0.6.15 RColorBrewer_1.1-2 checkmate_1.8.5
[19] qvalue_2.10.0 colorspace_1.3-2 R.oo_1.21.0
[22] GSEABase_1.40.1 XML_3.98-1.9 pkgconfig_2.0.1
[25] reactome.db_1.62.0 purrr_0.2.4 xtable_1.8-2
[28] GO.db_3.5.0 scales_0.5.0 gdata_2.18.0
[31] BiocParallel_1.12.0 tibble_1.4.2 annotate_1.56.2
[34] lazyeval_0.2.1 survival_2.41-3 magrittr_1.5
[37] memoise_1.1.0 R.methodsS3_1.7.1 hwriter_1.3.2
[40] tools_3.4.3 data.table_1.10.4-3 stringr_1.3.0
[43] munsell_0.4.3 compiler_3.4.3 caTools_1.17.1
[46] rlang_0.2.0 rappdirs_0.3.1 AnnotationForge_1.20.0
[49] gtable_0.2.0 DBI_0.8 R6_2.2.2
[52] gridExtra_2.3 graphite_1.24.1 bit_1.1-12
[55] fastmatch_1.1-0 fgsea_1.4.1 KernSmooth_2.23-15
[58] Rgraphviz_2.22.0 GOSemSim_2.4.1 stringi_1.1.7
[61] Rcpp_0.12.16 png_0.1-7

was any parameter set wrong?....
Thanks for your any guidance in advance~

Best,
Jianmei Zhong

Hi,l am Yudonglin from PUMC in china, your work is very good and I like it!
Reconstruction of complex single-cell trajectories using CellRouter | Nature Communications
https://www.nature.com/articles/s4 1467-018-03214-y#MOESM17
My problem is that I still have trouble running the example code in WINDOWS. I copy your code and run it in RStudio, yet "could not find function" is klling me. is there any chance you can solve that?Linux is not available for us, not to mention oracle java. If you can build a R package, maybe more user-friendly?
Thanks~

Hi,

I'm having an issue when I try to run the findPaths function. I'm following the tutorial with my own data, and up to that point everything works fine. When I get to

##Identify trajectories
cellrouter <- findPaths(cellrouter, column='seurat_clusters', libdir, paste(getwd()
, 'reports/CellRanger/files/paths', sep='/'), method="graph")

I get the following error for each trajectory (I copy here only 3 to 6 as an example):

-------------------Transition: 3.6  -----------------------
1) Computing flow network
Creating flow network...
Uncaught exception - null
java.lang.NullPointerException
	at cellrouter.InputOutput.getNetworkData(InputOutput.java:54)
	at cellrouter.CellRouter.createFlowNetwork(CellRouter.java:83)
	at cellrouter.CellRouter.runAnalysis(CellRouter.java:63)
	at cellrouter.CellRouter.main(CellRouter.java:244)

Can you give me any clue as to what could be causing the problem?

Hi, I met an error when I run processTrajectories.
The error is following:
Error in file(file, "rt") : cannot open the connection
In addition: Warning message:
In file(file, "rt") :
cannot open file 'C:/Users/Desktop/benthany/cellrouter-master/cellrouter-master/Myeloid_Progenitors/results/paths/6.1/Cells_FlowNetwork_all_paths.txt': No such file or directory
Called from: file(file, "rt")
Browse[1]>

Thank you~
Haichao

Hi,
Question 1)
Is there a way to plottrajectory without smoothening? I want to look at what the "raw" expression change trajectory looks like beneath the smoothed graph.

For example, plotting this gives me the "smoothed" curves for a sample of endothelial cells transitioning from arterial to venous.

genelist <- c("Pecam1", "Kdr", "Jag1")
plottrajectories(cellrouter, '8.9', genelist, rescale = TRUE, columns=1, width=5, height=2, filename='results/dynamics_curve.pdf')

http://127.0.0.1:33780/graphics/plot_zoom_png?width=467&height=314

Question 2)
Genes that should be relatively constant across a population of cells do not appear constant in the plot trajectories function.
For example, these genes (particularly Pecam1 and Flt1, "pan endothelial markers") should be relatively constant along this endothelial population, but instead they show extremely dramatic changes across the cell router trajectory:

http://127.0.0.1:33780/graphics/plot_zoom_png?width=467&height=314

http://127.0.0.1:33780/graphics/plot_zoom_png?width=467&height=314

What is happening so that they show such dramatic expression changes?

Question 3)
Is there a way to superimpose the cell router trajectory onto the TSNE? Or is there some other way to see what cells from where in the TSNE represent which stages along the cell router analysis?

#1 Which gene filtering is suited - m3drop, highly variable genes, highly dropout genes, highest PCA loading?
#2 Example Vignette starts from filtered data set - So what is the suitable input format for cellrouter - rawcounts / log2 counts / TPM / log2 TPM / FPKM / RPKM ?

Thank you for a very interesting tool for analyzing sc-data!

Trying to run through the tutorial I have come across an issue which seems to be related to java. At first, when running "findPaths" i got the error "No java runtime present, requesting install". Installation of Java Development Kit seemed to resolve this initial issue, however, now leaves me with the following, which i can't get my head around how to fix.

cellrouter <- findPaths(cellrouter, column='cluster', libdir, paste(getwd(), 'results/paths', sep='/'), method="graph")
-------------------Transition: 10.4 -----------------------

1. Computing flow network
   Error: Could not find or load main class cellrouter.CellRouter

Running findPaths creates the directories 10.4 (and remaining combinations) with the files cell_skins.txt, cell_sources.txt and CellRouter.sh, but not Cells_FlowNetwork_all_paths.txt which seems to be required for running processTrajectories afterwards.

Have you encountered this issue before and have any ideas how to resolve it?

Thank you very much,
Tobias

Hi,

I have already my cell clustered by seurat, now I want to use cell router for trajectory analysis? how that would be please? saying cellrouter@sampTab will help but I don't know how to use that for using my seurat clusters here

Thanks a lot for any help

Hello,
Congratulations for your recent work.
I was trying to complete your tutorial describing how to apply CellRouter to the mouse bone marrow single-cell RNA-sequencing dataset (Thank you very much for this tutorial, it is highly useful as a new user in R).
I was able to obtain the file "CellRouter_StemID.R", but just after this step, I started to have some troubles.

cellrouter <- processtrajectories(cellrouter, genes2use, path.rank=ranks[3],

•                               num.cells = 3, neighs = 1)
  

Error in processtrajectories(cellrouter, genes2use, path.rank = ranks[3], :
could not find function "processtrajectories"

Do you have an idea of this problem ? I am working with Windows
(English is not my native language, I apologize for that).

Thank you

Dear Developer,

I would like to ask if there is a a way of plotting multiple annotations (Cluster info, cell type, other ) from the metadata to the top of the heat map after doing DE analysis .. Like this one:

Thank you in advance!

Best,
Abdelrahman

Dear Colleuge,

I would like to ask about mean.p and mean.np in the differential expression analysis between populations and clusters. Can you please inform me what does they mean? and How have been calculated?

Best,
Abdelrahman

Hello,
In order to apply CellRouter to my own datasets in the future, I would like to try to complete (and master) your tutorial with the mouse bone marrow single-cell RNA-sequencing dataset (I am a new user in R).
You mentionned at the beginning of the tutorial that "CellRouter assumes that you are providing a gene expression matrix with genes as rows and single-cells as columns." I understand that these are the normalized and filtered_expression files provided.

However, I was wondering how to obtain in the future the file named "matrix.R" that you use in "Create Cell Router" part. It is a matrix with two columns and single cells in rownames.
You mentioned to rename these columns as tSNE1 and tSNE2, so I assumed that this matrix could come from a "tsne analysis", is it correct or am I wrong ?

I tried to do this with filtered_expression files using
fdata1<-t(fdata) # transposition
"test<-Rtsne(as.matrix(fdata1), dims=2, check_duplicates=FALSE, pca=T, perplexity=30, theta=0.5, max_iter = 5000)
matrix <- as.data.frame(test$Y) # matrix 2 columns 1007 rows
Using this matrix I obtained a different graph, "similar" but different.

could you indicate me what are the main steps to obtain this "matrix" ?
(I apologize for my English)

Thank you