ERROR ~ Error executing process > 'bambu_EM'

[fzm311]

Hello, could you help me check why my program isn't running? To use bambu-singlecell-spatial on the HPC platform, I cloned the main branch and pulled the image as bambusc_beta1.2.sif using Singularity. Then, when running the test case, an error occurred. Below are the commands I ran and the error message.

**(base) fzm@m:~/home/bambu-singlecell-spatial$ nextflow run . --reads /home/fzm/home/bambu-singlecell-spatial/examples/reads_chr9_1_1000000.fastq.gz --genome /home/fzm/home/bambu-singlecell-spatial/examples/Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa --annotation /home/fzm/home/bambu-singlecell-spatial/examples/Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf --chemistry 10x5v2 --ncore 4 --outdir output -with-singularity '/home/fzm/home/bambusc_beta1.2.sif'
Nextflow 25.10.4 is available - Please consider updating your version to it

N E X T F L O W ~ version 25.10.3

Launching ./main.nf [amazing_colden] DSL2 - revision: 5dc06a459c

executor > local (4)
[ff/0a7aa7] flexiplex (1) [100%] 1 of 1 ✔
[0c/c09d3d] minimap (1) [100%] 1 of 1 ✔
[72/ecbe40] bambu [100%] 1 of 1 ✔
[7d/b138ba] bambu_EM [ 0%] 0 of 1
ERROR ~ Error executing process > 'bambu_EM'

Caused by:
Process bambu_EM terminated with an error exit status (1)

Command executed:

#!/usr/bin/env Rscript
#.libPaths("/usr/local/lib/R/site-library")
library(devtools)
if("bambu" == "bambu") {
load_all("/mnt/software/bambu")
} else {
load_all("bambu")
}
if(".txt" %in% "combined"){runName = readLines("combined")
} else{runName = "combined"}

extendedAnno <- readRDS("Bambu__extended_annotations.rds")
quantDatas = readRDS("Bambu__quantData.rds")
clusters = readRDS("Bambu__clusters.rds")
degBias = TRUE
if(is.null(clusters)){degBias = FALSE}

se = bambu( reads = "test.rds",
annotations = extendedAnno,
genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa",
quantData = quantDatas,
assignDist = FALSE,
ncore = 4,
discovery = FALSE,
quant = TRUE,
demultiplexed = TRUE,
verbose = FALSE,
opt.em = list(degradationBias = degBias),
clusters = clusters)

saveRDS(se, paste0(runName, "_se.rds"))
writeBambuOutput(se, path = ".", prefix = paste0(runName, "EM"),outputExtendedAnno = FALSE,
outputAll = FALSE, outputBambuModels = FALSE, outputNovelOnly = FALSE)

Command exit status:
1

Command output:
(empty)

Command error:
I, expand.grid, unname

Loading required package: IRanges
Loading required package: GenomeInfoDb
Loading required package: Biobase
Welcome to Bioconductor

  Vignettes contain introductory material; view with
  'browseVignettes()'. To cite Bioconductor, see
  'citation("Biobase")', and for packages 'citation("pkgname")'.

Attaching package: 'Biobase'

The following object is masked from 'package:MatrixGenerics':

  rowMedians

The following objects are masked from 'package:matrixStats':

  anyMissing, rowMedians

Loading required package: BSgenome
Loading required package: Biostrings
executor > local (4)
[ff/0a7aa7] flexiplex (1) [100%] 1 of 1 ✔
[0c/c09d3d] minimap (1) [100%] 1 of 1 ✔
[72/ecbe40] bambu [100%] 1 of 1 ✔
[7d/b138ba] bambu_EM [ 0%] 0 of 1 ✘
ERROR ~ Error executing process > 'bambu_EM'

Caused by:
Process bambu_EM terminated with an error exit status (1)

Command executed:

#!/usr/bin/env Rscript
#.libPaths("/usr/local/lib/R/site-library")
library(devtools)
if("bambu" == "bambu") {
load_all("/mnt/software/bambu")
} else {
load_all("bambu")
}
if(".txt" %in% "combined"){runName = readLines("combined")
} else{runName = "combined"}

extendedAnno <- readRDS("Bambu__extended_annotations.rds")
quantDatas = readRDS("Bambu__quantData.rds")
clusters = readRDS("Bambu__clusters.rds")
degBias = TRUE
if(is.null(clusters)){degBias = FALSE}

se = bambu( reads = "test.rds",
annotations = extendedAnno,
genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa",
quantData = quantDatas,
assignDist = FALSE,
ncore = 4,
discovery = FALSE,
quant = TRUE,
demultiplexed = TRUE,
verbose = FALSE,
opt.em = list(degradationBias = degBias),
clusters = clusters)

saveRDS(se, paste0(runName, "_se.rds"))
writeBambuOutput(se, path = ".", prefix = paste0(runName, "EM"),outputExtendedAnno = FALSE,
outputAll = FALSE, outputBambuModels = FALSE, outputNovelOnly = FALSE)

Command exit status:
1

Command output:
(empty)

Command error:
I, expand.grid, unname

Loading required package: IRanges
Loading required package: GenomeInfoDb
Loading required package: Biobase
Welcome to Bioconductor

  Vignettes contain introductory material; view with
  'browseVignettes()'. To cite Bioconductor, see
  'citation("Biobase")', and for packages 'citation("pkgname")'.

Attaching package: 'Biobase'

The following object is masked from 'package:MatrixGenerics':

  rowMedians

The following objects are masked from 'package:matrixStats':

  anyMissing, rowMedians

Loading required package: BSgenome
Loading required package: Biostrings
Loading required package: XVector

Attaching package: 'Biostrings'

The following object is masked from 'package:base':

  strsplit

Loading required package: BiocIO
Loading required package: rtracklayer

Attaching package: 'rtracklayer'

The following object is masked from 'package:BiocIO':

  FileForFormat

Running Bambu-v3.9.0
WARNING - If you change the number of cores (ncore) between Bambu runs and there is no progress please restart your R session to resolve the issue that originates from the XGboost package.
--- Start isoform EM quantification ---
Error: BiocParallel errors
1 remote errors, element index: 1
0 unevaluated and other errors
first remote error:
Error in (function (cond) : error in evaluating the argument 'obj' in selecting a method for function 'unname': object of type 'S4' is not subsettable
Execution halted

Work dir:
/home/fzm/home/bambu-singlecell-spatial/work/7d/b138ba9e637df0d1cbb4f9a93f8d9d

Container:
/home/fzm/home/bambusc_beta1.2.sif

Tip: view the complete command output by changing to the process work dir and entering the command cat .command.out

-- Check '.nextflow.log' file for details
**

[fzm311]

When I used the command from the README document, it even got stuck at the bambu step. Below is the corresponding error output.

(base) fzm@m:~/home/bambu-singlecell-spatial$ nextflow run GoekeLab/bambu-singlecell-spatial --reads /home/fzm/home/bambu-singlecell-
spatial/examples/reads_chr9_1_1000000.fastq.gz --genome /home/fzm/home/bambu-singlecell-spatial/examples/Homo_sapiens.GRCh38.dna_sm.p
rimary_assembly_chr9_1_1000000.fa --annotation /home/fzm/home/bambu-singlecell-spatial/examples/Homo_sapiens.GRCh38.91_chr9_1_1000000
.gtf --chemistry 10x5v2 --ncore 4 --outdir output -with-singularity lingminhao/bambusc:beta1.2
Nextflow 25.10.4 is available - Please consider updating your version to it

N E X T F L O W ~ version 25.10.3

Launching https://github.com/GoekeLab/bambu-singlecell-spatial [fervent_swartz] DSL2 - revision: 38eb5de [main]

executor > local (3)
[7c/8e5caa] flexiplex (1) [100%] 1 of 1 ✔
[4e/92cee6] minimap (1) [100%] 1 of 1 ✔
[13/64525a] bambu [ 0%] 0 of 1
[- ] bambu_EM -
ERROR ~ Error executing process > 'bambu'

Caused by:
Process bambu terminated with an error exit status (1)

Command executed:

#!/usr/bin/env Rscript
#.libPaths("/usr/local/lib/R/site-library")

 samples = "[/home/fzm/home/bambu-singlecell-spatial/work/4e/92cee654b3188fd9343b0c6adf18dc/Bambu.demultiplexed.bam]"
 samples = gsub("[][]","", gsub(' ','', samples))
 samples = unlist(strsplit(samples, ','))

 ids = "[Bambu]"
 ids = gsub("[][]","", gsub(' ','', ids))
 ids = unlist(strsplit(ids, ','))
 if(length(ids)>1){runName = "combined_"
 }else{runName = paste0(ids, "_")}

 if(file.exists("TRUE")){
     x = gsub("[][]","",gsub(' ','', "TRUE"))
     barcode_maps = unlist(strsplit(x, ','))
 } else {
     barcode_maps = TRUE
 }

 library(devtools)
 if("bambu" == "bambu") {
     load_all("/mnt/software/bambu")
 } else {
     load_all("bambu")
 }

 spatial = "FALSE"
 if(spatial == "FALSE"){spatial = NULL}

annotations <- prepareAnnotations("Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf")

Transcript discovery and generate readGrgList for each cell

 readClassFile = bambu(reads = samples, annotations = annotations, genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa",
     ncore = 4, discovery = FALSE, quant = FALSE, demultiplexed = barcode_maps,
     verbose = FALSE, assignDist = FALSE, processByChromosome = as.logical("TRUE"),
     processByBam = as.logical("FALSE"), yieldSize = 10000000,
     sampleNames = ids, cleanReads = as.logical("TRUE"), dedupUMI = as.logical("TRUE"))
 saveRDS(readClassFile, paste0(runName, "_readClassFile.rds"))
 if(isFALSE(NULL)){
     extendedAnno = bambu(reads = readClassFile, annotations = annotations, genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", ncore = 4, discovery = TRUE, quant = FALSE, demultiplexed = TRUE, verbose = FALSE, assignDist = FALSE)
 } else{
     extendedAnno = bambu(reads = readClassFile, annotations = annotations, genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", ncore = 4, discovery = TRUE, quant = FALSE, demultiplexed = TRUE, verbose = FALSE, assignDist = FALSE, NDR = NULL)
 }
 saveRDS(extendedAnno, paste0(runName, "_extended_annotations.rds"))
 rm(annotations)
 se = bambu(reads = readClassFile, annotations = extendedAnno, genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", ncore = 4, discovery = FALSE, quant = FALSE, demultiplexed = TRUE, verbose = FALSE, opt.em = list(degradationBias = FALSE), assignDist = TRUE, spatial = spatial)
 saveRDS(se, paste0(runName, "_quantData.rds"))
 for(se.x in se){
     if(as.logical("FALSE")){
         writeBambuOutput(se.x, '.', prefix = metadata(se.x)$sampleNames)
     } else{
         writeBambuOutput(se.x, '.', prefix = "combined_")
     }

 }
 #writeBambuOutput(do.call(cbind, se), '.')
 #write(runName, "runName.txt")

 #if no clustering provided, automatically cluster
 if("auto" == "auto"){
     clusters = list()
     cellMixs = list()
     source("/home/fzm/.nextflow/assets/GoekeLab/bambu-singlecell-spatial/utilityFunctions.R")
     for(quantData in se){
         quantData.gene = transcriptToGeneExpression(quantData)
         for(sample in unique(colData(quantData)$sampleName)){
             i = which(colData(quantData)$sampleName == sample)
             counts = assays(quantData.gene)$counts[,i]
             cellMix = clusterCells(counts, resolution = 0.8)
             x = setNames(names(cellMix@active.ident), cellMix@active.ident)
             names(x) = paste0(sample,"_",names(x))
             clusters = c(clusters, splitAsList(unname(x), names(x)))
             cellMixs = c(cellMixs, cellMix)
         }
     }
     saveRDS(cellMixs, paste0(runName, "_cellMixs.rds"))
 }
 if("auto" == "none"){
     clusters = NULL
 }
 saveRDS(clusters, paste0(runName, "_clusters.rds"))

Command exit status:
1

Command output:
https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:02] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:02] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:03] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:06] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:06] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[1] "counts"

Command error:
for more details about differences between saving model and serializing.

[21:39:03] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

Number of Read Classes - 92
[21:39:06] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:06] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
executor > local (3)
[7c/8e5caa] flexiplex (1) [100%] 1 of 1 ✔
[4e/92cee6] minimap (1) [100%] 1 of 1 ✔
[13/64525a] bambu [ 0%] 0 of 1 ✘
[- ] bambu_EM -
ERROR ~ Error executing process > 'bambu'

Caused by:
Process bambu terminated with an error exit status (1)

Command executed:

#!/usr/bin/env Rscript
#.libPaths("/usr/local/lib/R/site-library")

 samples = "[/home/fzm/home/bambu-singlecell-spatial/work/4e/92cee654b3188fd9343b0c6adf18dc/Bambu.demultiplexed.bam]"
 samples = gsub("[][]","", gsub(' ','', samples))
 samples = unlist(strsplit(samples, ','))

 ids = "[Bambu]"
 ids = gsub("[][]","", gsub(' ','', ids))
 ids = unlist(strsplit(ids, ','))
 if(length(ids)>1){runName = "combined_"
 }else{runName = paste0(ids, "_")}

 if(file.exists("TRUE")){
     x = gsub("[][]","",gsub(' ','', "TRUE"))
     barcode_maps = unlist(strsplit(x, ','))
 } else {
     barcode_maps = TRUE
 }

 library(devtools)
 if("bambu" == "bambu") {
     load_all("/mnt/software/bambu")
 } else {
     load_all("bambu")
 }

 spatial = "FALSE"
 if(spatial == "FALSE"){spatial = NULL}

annotations <- prepareAnnotations("Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf")

Transcript discovery and generate readGrgList for each cell

 readClassFile = bambu(reads = samples, annotations = annotations, genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa",
     ncore = 4, discovery = FALSE, quant = FALSE, demultiplexed = barcode_maps,
     verbose = FALSE, assignDist = FALSE, processByChromosome = as.logical("TRUE"),
     processByBam = as.logical("FALSE"), yieldSize = 10000000,
     sampleNames = ids, cleanReads = as.logical("TRUE"), dedupUMI = as.logical("TRUE"))
 saveRDS(readClassFile, paste0(runName, "_readClassFile.rds"))
 if(isFALSE(NULL)){
     extendedAnno = bambu(reads = readClassFile, annotations = annotations, genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", ncore = 4, discovery = TRUE, quant = FALSE, demultiplexed = TRUE, verbose = FALSE, assignDist = FALSE)
 } else{
     extendedAnno = bambu(reads = readClassFile, annotations = annotations, genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", ncore = 4, discovery = TRUE, quant = FALSE, demultiplexed = TRUE, verbose = FALSE, assignDist = FALSE, NDR = NULL)
 }
 saveRDS(extendedAnno, paste0(runName, "_extended_annotations.rds"))
 rm(annotations)
 se = bambu(reads = readClassFile, annotations = extendedAnno, genome = "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", ncore = 4, discovery = FALSE, quant = FALSE, demultiplexed = TRUE, verbose = FALSE, opt.em = list(degradationBias = FALSE), assignDist = TRUE, spatial = spatial)
 saveRDS(se, paste0(runName, "_quantData.rds"))
 for(se.x in se){
     if(as.logical("FALSE")){
         writeBambuOutput(se.x, '.', prefix = metadata(se.x)$sampleNames)
     } else{
         writeBambuOutput(se.x, '.', prefix = "combined_")
     }

 }
 #writeBambuOutput(do.call(cbind, se), '.')
 #write(runName, "runName.txt")

 #if no clustering provided, automatically cluster
 if("auto" == "auto"){
     clusters = list()
     cellMixs = list()
     source("/home/fzm/.nextflow/assets/GoekeLab/bambu-singlecell-spatial/utilityFunctions.R")
     for(quantData in se){
         quantData.gene = transcriptToGeneExpression(quantData)
         for(sample in unique(colData(quantData)$sampleName)){
             i = which(colData(quantData)$sampleName == sample)
             counts = assays(quantData.gene)$counts[,i]
             cellMix = clusterCells(counts, resolution = 0.8)
             x = setNames(names(cellMix@active.ident), cellMix@active.ident)
             names(x) = paste0(sample,"_",names(x))
             clusters = c(clusters, splitAsList(unname(x), names(x)))
             cellMixs = c(cellMixs, cellMix)
         }
     }
     saveRDS(cellMixs, paste0(runName, "_cellMixs.rds"))
 }
 if("auto" == "none"){
     clusters = NULL
 }
 saveRDS(clusters, paste0(runName, "_clusters.rds"))

Command exit status:
1

Command output:
https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:02] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:02] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:03] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:06] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:06] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[1] "counts"

Command error:
for more details about differences between saving model and serializing.

[21:39:03] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

Number of Read Classes - 92
[21:39:06] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

[21:39:06] WARNING: src/learner.cc:553:
If you are loading a serialized model (like pickle in Python, RDS in R) generated by
older XGBoost, please export the model by calling Booster.save_model from that version
first, then load it back in current version. See:

  https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

for more details about differences between saving model and serializing.

Running Bambu-v3.9.0
WARNING - If you change the number of cores (ncore) between Bambu runs and there is no progress please restart your R session to resolve the issue that originates from the XGboost package.
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
Using a novel discovery rate (NDR) of: 0
Novel transcripts detected: 0
Novel genes detected: 0
Low confidence transcripts excluded: 21
Running Bambu-v3.9.0
WARNING - If you change the number of cores (ncore) between Bambu runs and there is no progress please restart your R session to resolve the issue that originates from the XGboost package.
--- Start calculating equivilance classes ---
[1] "counts"
Error in file(filename, "r", encoding = encoding) :
cannot open the connection
Calls: source -> file
In addition: Warning message:
In file(filename, "r", encoding = encoding) :
cannot open file '/home/fzm/.nextflow/assets/GoekeLab/bambu-singlecell-spatial/utilityFunctions.R': No such file or directory
Execution halted

Work dir:
/home/fzm/home/bambu-singlecell-spatial/work/13/64525a0951e737d2cb5fc55bb6b8c0

Container:
/home/fzm/home/bambu-singlecell-spatial/work/singularity/lingminhao-bambusc-beta1.2.img

Tip: view the complete command output by changing to the process work dir and entering the command cat .command.out

-- Check '.nextflow.log' file for details