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Error in processTrajectories #25

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cjpark85 opened this issue May 8, 2019 · 8 comments
Open

Error in processTrajectories #25

cjpark85 opened this issue May 8, 2019 · 8 comments

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@cjpark85
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cjpark85 commented May 8, 2019 •
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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") :
@edroaldo
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edroaldo commented May 10, 2019 via email

@cjpark85
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cjpark85 commented May 10, 2019 •
edited
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I attached my script as below. I increased K in buildKNN, but it doesn't work.

data_total <- as.data.frame(as.matrix(GetAssayData(object =combined, slot = "counts")))
cellrouter <- CellRouter(rawdata=as.data.frame(data_total), min.cells=0, min.genes=0)
[1] "Initializing CellRouter object"
metadata_total <- combined@meta.data
cellrouter <- addInfo(cellrouter, metadata = metadata_total, colname = 'seurat_clusters', metadata.column='integrated_snn_res.0.5')
cellrouter <- addInfo(cellrouter, metadata=metadata_total, colname = 'seurat_clusters', metadata.column = 'integrated_snn_res.0.5')
cellrouter <- addInfo(cellrouter, metadata=metadata_total, colname = 'res.0.5', metadata.column = 'integrated_snn_res.0.5')
cellrouter <- addInfo(cellrouter, metadata=metadata_total, colname = 'group', metadata.column = 'geno')
class(cellrouter@ndata)
[1] "data.frame"
cellrouter <- Normalize(cellrouter)
cellrouter <- scaleData(cellrouter)
cellrouter <- computePCA(cellrouter, genes.use=rownames(cellrouter@ndata), num.pcs = 50, seed=42)
plot(cellrouter@pca$sdev, xlab='PC', ylab='Standard deviation of PC')
cellrouter <- computeTSNE(cellrouter, num.pcs = 12, seed=42, max_iter = 1000)
cellrouter <- customSpace(cellrouter, cellrouter@tsne$cell.embeddings)
dim(cellrouter@rawdata)
[1] 0 0
dim(cellrouter@ndata)
[1] 21355 6078
dim(cellrouter@sampTab)
[1] 6078 10
cellrouter@ndata[1:5,1:5]
AAACCCAAGGATACCG_1 AAACCCACAGAGTTGG_1 AAACCCAGTATGCAAA_1 AAACCCAGTCTTTCAT_1 AAACCCATCGCTCTCA_1
Xkr4 0 0 0 0.000000 0
Gm37686 0 0 0 0.000000 0
Gm38148 0 0 0 0.000000 0
Gm19938 0 0 0 0.000000 0
Sox17 0 0 0 1.231911 0

plotReducedDimension(cellrouter, reduction.type = 'tsne', dims.use = c(1,2), annotation = "seurat_clusters", annotation.color = 'seurat_clusters_color', showlabels = TRUE, 4.5, 3.5, filename='D:/Single-cell_ovary_hCG/6h_050719/tSNE_total1.pdf')
cellrouter <- computeTSNE(cellrouter, num.pcs = 20, seed=42, max_iter = 1000)
cellrouter <- customSpace(cellrouter, cellrouter@tsne$cell.embeddings)
slotNames(cellrouter@ndata)
[1] ".Data" "names" "row.names" ".S3Class"
cellrouter@sampTab[1:5,]
sample_id nGene nUMI conditions seurat_clusters seurat_clusters_color res.0.5 res.0.5_color group group_color
AAACCCAAGGATACCG_1 AAACCCAAGGATACCG_1 5057 21978 AAACCCAAGGATACCG_1 8 #A6CEE3 8 #A6CEE3 WT #A6CEE3
AAACCCACAGAGTTGG_1 AAACCCACAGAGTTGG_1 6783 57925 AAACCCACAGAGTTGG_1 10 #A6CEE3 10 #A6CEE3 WT #A6CEE3
AAACCCAGTATGCAAA_1 AAACCCAGTATGCAAA_1 2825 6567 AAACCCAGTATGCAAA_1 3 #A6CEE3 3 #A6CEE3 WT #A6CEE3
AAACCCAGTCTTTCAT_1 AAACCCAGTCTTTCAT_1 3789 12357 AAACCCAGTCTTTCAT_1 7 #A6CEE3 7 #A6CEE3 WT #A6CEE3
AAACCCATCGCTCTCA_1 AAACCCATCGCTCTCA_1 5880 29607 AAACCCATCGCTCTCA_1 3 #A6CEE3 3 #A6CEE3 WT #A6CEE3

markers <- findSignatures(cellrouter, column = "seurat_clusters", pos.only = TRUE, fc.threshold = 0.5)
Calculating fold changes...
[1] "discovering subpopulation-specific gene signatures"
cluster 8
[1] 136
cluster 10
[1] 159
cluster 3
[1] 161
cluster 7
[1] 296
cluster 5
[1] 158
cluster 0
[1] 211
cluster 4
[1] 84
cluster 6
[1] 14
cluster 2
[1] 63
cluster 9
[1] 188
cluster 16
[1] 346
cluster 1
[1] 155
cluster 14
[1] 142
cluster 15
[1] 216
cluster 11
[1] 98
cluster 13
[1] 176
cluster 12
[1] 258
cluster 17
[1] 312
[1] "finding subpopulation markers"
top10 <- markers %>% group_by(population) %>% top_n(10, fc)
filename <- 'results/path/heatmap_top_10_genes_sorted_populations_total.png'
plotSignaturesHeatmap(cellrouter, markers = top10, column.ann = 'seurat_clusters', column.color = 'seurat_clusters_color', threshold = 2, width = 7, height = 7, filename=filename)
[1] "here"

0 1 10 11 12 13 14 15 16 17 2 3 4 5 6 7 8 9
1031 737 214 195 151 150 94 72 42 21 681 573 503 398 394 331 264 227
used (Mb) gc trigger (Mb) max used (Mb)
Ncells 3408012 182.1 9299112 496.7 9299112 496.7
Vcells 1346928212 10276.3 2434345478 18572.6 2434345363 18572.6

Identification of Trajectories between clusters

cellrouter <- buildKNN(cellrouter, k = 10, column.ann = 'seurat_clusters', num.pcs = 20, sim.type = 'jaccard')
[1] "building k-nearest neighbors graph"
[1] "updating CellRouter object"
Warning message:
In write.graph.gml(graph, file, ...) :
At foreign.c:2616 :A boolean graph attribute was converted to numeric
plotKNN(cellrouter, reduction.type = 'tsne', column.ann = 'seurat_clusters', column.color = 'seurat_clusters_color', width = 5, height = 4, filename='results/path/knn_tsne_original_clusters_total.pdf')
Loading required package: sna
Loading required package: statnet.common

Attaching package: ‘statnet.common’

The following object is masked from ‘package:bit64’:

order

The following object is masked from ‘package:BiocGenerics’:

order

The following object is masked from ‘package:base’:

order

Loading required package: network
network: Classes for Relational Data
Version 1.15 created on 2019-04-01.
copyright (c) 2005, Carter T. Butts, University of California-Irvine
Mark S. Handcock, University of California -- Los Angeles
David R. Hunter, Penn State University
Martina Morris, University of Washington
Skye Bender-deMoll, University of Washington
For citation information, type citation("network").
Type help("network-package") to get started.

Attaching package: ‘network’

The following objects are masked from ‘package:igraph’:

%c%, %s%, add.edges, add.vertices, delete.edges, delete.vertices, get.edge.attribute, get.edges, get.vertex.attribute, is.bipartite,
is.directed, list.edge.attributes, list.vertex.attributes, set.edge.attribute, set.vertex.attribute

The following object is masked from ‘package:plyr’:

is.discrete

sna: Tools for Social Network Analysis
Version 2.4 created on 2016-07-23.
copyright (c) 2005, Carter T. Butts, University of California-Irvine
For citation information, type citation("sna").
Type help(package="sna") to get started.

Attaching package: ‘sna’

The following object is masked from ‘package:graph’:

degree

The following objects are masked from ‘package:igraph’:

betweenness, bonpow, closeness, components, degree, dyad.census, evcent, hierarchy, is.connected, neighborhood, triad.census

The following object is masked from ‘package:bit’:

nties

Warning messages:
1: package ‘sna’ was built under R version 3.5.3
2: package ‘statnet.common’ was built under R version 3.5.3
3: package ‘network’ was built under R version 3.5.3

cellrouter <- buildKNN(cellrouter, k = 10, column.ann = 'population', num.pcs = 20, sim.type = 'jaccard')
Show Traceback

Rerun with Debug
Error in base::order(..., na.last = na.last, decreasing = decreasing) :
argument 1 is not a vector

filename <- "results/paths/cell_edge_weighted_network.txt"
write.table(cellrouter@graph$edges, file=filename, sep='\t', row.names=FALSE, col.names = FALSE, quote=FALSE)
sources <- c('0')
targets <- setdiff(as.vector(cellrouter@sampTab$seurat_clusters), sources)
methods <- c("euclidean", "maximum", "manhattan","canberra","binary", 'graph') #graph for distances in KNN
cellrouter <- findPaths(cellrouter, column='seurat_clusters', libdir, paste(getwd(), 'results/paths', sep='/'), method="graph")
-------------------Transition: 0.8 -----------------------
-------------------Transition: 0.10 -----------------------
-------------------Transition: 0.3 -----------------------
-------------------Transition: 0.7 -----------------------
-------------------Transition: 0.5 -----------------------
-------------------Transition: 0.4 -----------------------
-------------------Transition: 0.6 -----------------------
-------------------Transition: 0.2 -----------------------
-------------------Transition: 0.9 -----------------------
-------------------Transition: 0.16 -----------------------
-------------------Transition: 0.1 -----------------------
-------------------Transition: 0.14 -----------------------
-------------------Transition: 0.15 -----------------------
-------------------Transition: 0.11 -----------------------
-------------------Transition: 0.13 -----------------------
-------------------Transition: 0.12 -----------------------
-------------------Transition: 0.17 -----------------------
ranks <- c('path_cost', 'path_flow', 'rank', 'length')
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") :

cellrouter <- buildKNN(cellrouter, k = 20, column.ann = 'seurat_clusters', num.pcs = 20, sim.type = 'jaccard')
[1] "building k-nearest neighbors graph"
[1] "updating CellRouter object"
Warning message:
In write.graph.gml(graph, file, ...) :
At foreign.c:2616 :A boolean graph attribute was converted to numeric
plotKNN(cellrouter, reduction.type = 'tsne', column.ann = 'seurat_clusters', column.color = 'seurat_clusters_color', width = 5, height = 4, filename='D:/results/paths/knn_tsne_original_clusters_total1.pdf')

cellrouter <- buildKNN(cellrouter, k = 20, column.ann = 'population', num.pcs = 20, sim.type = 'jaccard')
Show Traceback

Rerun with Debug
Error in base::order(..., na.last = na.last, decreasing = decreasing) :
argument 1 is not a vector > plotKNN(cellrouter, reduction.type = 'tsne', column.ann = 'population', column.color = 'colors', width = 5, height = 4, filename='results/paths/knn_tsne_CellRouter_clusters1.pdf')
Show Traceback

Rerun with Debug
Error: Insufficient values in manual scale. 18 needed but only 0 provided.

filename <- "results/paths/cell_edge_weighted_network.txt"
write.table(cellrouter@graph$edges, file=filename, sep='\t', row.names=FALSE, col.names = FALSE, quote=FALSE)
sources <- c('0')
targets <- setdiff(as.vector(cellrouter@sampTab$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: 0.8 -----------------------
-------------------Transition: 0.10 -----------------------
-------------------Transition: 0.3 -----------------------
-------------------Transition: 0.7 -----------------------
-------------------Transition: 0.5 -----------------------
-------------------Transition: 0.4 -----------------------
-------------------Transition: 0.6 -----------------------
-------------------Transition: 0.2 -----------------------
-------------------Transition: 0.9 -----------------------
-------------------Transition: 0.16 -----------------------
-------------------Transition: 0.1 -----------------------
-------------------Transition: 0.14 -----------------------
-------------------Transition: 0.15 -----------------------
-------------------Transition: 0.11 -----------------------
-------------------Transition: 0.13 -----------------------
-------------------Transition: 0.12 -----------------------
-------------------Transition: 0.17 -----------------------

ranks <- c('path_cost', 'path_flow', 'rank', 'length')
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") :

@cjpark85
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Hi,

Today, I tried again and got this error msg. I don't know what is 'file(file, "rt")'. Also, I checked '~/results/path/8.2/' directory, but I couldn't find 'Cells_FlowNetwork_all_paths.txt' file. Is this generated by 'processTrajectories' function? How can I solve this problem?

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") :
cannot open file '~/results/paths/8.2/Cells_FlowNetwork_all_paths.txt': No such file or directory

@edroaldo
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edroaldo commented May 13, 2019 via email

@cjpark85
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In the meantime, I'll keep trying. Thank you for your reply.

@cjpark85
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cjpark85 commented Jun 5, 2019

Hello edroaldo,
I'm still struggling with this issue. I'm sorry. I need some help.

@edroaldo
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edroaldo commented Jun 5, 2019 via email

@cjpark85
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Thank you for reviewing this script. I ran again according to your suggestion, but I can see same error. I'm sorry to keep bothering you. Actually, I can't find the file 'Cells_FlowNetwork_all_paths.txt'.

> # Identification of Trajectories between clusters 
> cellrouter <- buildKNN(cellrouter, k = 15, column.ann = 'seurat_clusters', num.pcs = 25, sim.type = 'jaccard')
[1] "building k-nearest neighbors graph"
[1] "updating CellRouter object"
Warning message:
In write.graph.gml(graph, file, ...) :
  At foreign.c:2616 :A boolean graph attribute was converted to numeric
> plotKNN(cellrouter, reduction.type = 'tsne', column.ann = 'seurat_clusters', 
+         column.color = 'seurat_clusters_color', width = 5, height = 4, 
+         filename='D:/cellrouter-master/CellRouter/results/knn_tsne_original_clusters.pdf')
> 
> setwd("D:/cellrouter-master/CellRouter")
> 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)
> sources <- c('2', '4')
> targets <- c('3', '7')
> methods <- c("euclidean", "maximum", "manhattan","canberra","binary", 'graph')
> ##Identify trajectories
> cellrouter <- findPaths(cellrouter, column='seurat_clusters', libdir, paste(getwd(), 'results/paths', sep='/'), method="graph")
-------------------Transition: 2.3  -----------------------
-------------------Transition: 2.7  -----------------------
-------------------Transition: 4.3  -----------------------
-------------------Transition: 4.7  -----------------------
> 
> ranks <- c('path_cost', 'path_flow', 'rank', 'length')
> 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:
  cannot open file 'D:/cellrouter-master/CellRouter/results/paths/2.3/Cells_FlowNetwork_all_paths.txt': No such file or directory

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@edroaldo @cjpark85