'strsplit': object 'GE' not foundBUG

Hi, We're getting the below error, near the end of the pipeline. Any idea what the issue is? Thanks

 You provided these parameters:
 YAML file: RNAseq_4-17-23.yaml
 zUMIs directory:       /gpfs/data/lab/bin/zUMIs
 STAR executable        /gpfs/data/lab/bin/STAR/STAR
 samtools executable        samtools
 pigz executable        pigz
 Rscript executable     Rscript
 RAM limit:   140
 zUMIs version 2.9.7e 

Thu Apr 27 23:21:39 EDT 2023
WARNING: The STAR version used for mapping is 2.7.7a and the STAR index was created using the version 2.7.4a. This may lead to an error while mapping. If you encounter any errors at the mapping stage, please make sure to create the STAR index using STAR 2.7.7a.
Filtering...
Fri Apr 28 00:10:51 EDT 2023
[1] " reads were assigned to barcodes that do not correspond to intact cells."
Mapping...
[1] "2023-04-28 00:13:06 EDT"
Apr 28 00:13:07 ..... started STAR run
Apr 28 00:13:07 ..... loading genome
Apr 28 00:13:07 ..... started STAR run
Apr 28 00:13:08 ..... loading genome
Apr 28 00:13:07 ..... started STAR run
Apr 28 00:13:08 ..... loading genome
Apr 28 00:13:07 ..... started STAR run
Apr 28 00:13:08 ..... loading genome
Apr 28 00:13:07 ..... started STAR run
Apr 28 00:13:08 ..... loading genome
Apr 28 00:15:26 ..... processing annotations GTF
Apr 28 00:15:26 ..... processing annotations GTF
Apr 28 00:15:26 ..... processing annotations GTF
Apr 28 00:15:26 ..... processing annotations GTF
Apr 28 00:15:26 ..... processing annotations GTF
Apr 28 00:17:44 ..... inserting junctions into the genome indices
Apr 28 00:17:44 ..... inserting junctions into the genome indices
Apr 28 00:17:44 ..... inserting junctions into the genome indices
Apr 28 00:17:44 ..... inserting junctions into the genome indices
Apr 28 00:17:44 ..... inserting junctions into the genome indices
Apr 28 00:28:59 ..... started mapping
Apr 28 00:28:59 ..... started mapping
Apr 28 00:28:59 ..... started mapping
Apr 28 00:28:59 ..... started mapping
Apr 28 00:28:59 ..... started mapping
Apr 28 00:50:52 ..... finished mapping
Apr 28 00:50:54 ..... finished successfully
Apr 28 02:01:23 ..... finished mapping
Apr 28 02:01:25 ..... finished successfully
Apr 28 02:06:11 ..... finished mapping
Apr 28 02:06:42 ..... finished successfully
Apr 28 02:08:40 ..... finished mapping
Apr 28 02:09:18 ..... finished successfully
Apr 28 02:09:19 ..... finished mapping
Apr 28 02:09:21 ..... finished successfully
Fri Apr 28 02:12:00 EDT 2023
Counting...
[1] "2023-04-28 02:12:16 EDT"
[1] "2.1e+08 Reads per chunk"
[1] "Loading reference annotation from:"
[1] "/gpfs/data/lab/projects/seq/analysis/RNAseq_4-17-23/RNAseq_4-17-23.final_annot.gtf"
[1] "Annotation loaded!"
[1] "Assigning reads to features (ex)"

        ==========     _____ _    _ ____  _____  ______          _____  
        =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
          =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
            ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
              ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
        ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
       Rsubread 1.32.4

//========================== featureCounts setting ===========================\\
||                                                                            ||
||             Input files : 1 BAM file                                       ||
||                           P RNAseq_4-17-23.filtered.tagged.Aligned.out ... ||
||                                                                            ||
||              Annotation : R data.frame                                     ||
||      Assignment details : <input_file>.featureCounts.bam                   ||
||                      (Note that files are saved to the output directory)   ||
||                                                                            ||
||      Dir for temp files : .                                                ||
||                 Threads : 16                                               ||
||                   Level : meta-feature level                               ||
||              Paired-end : yes                                              ||
||      Multimapping reads : counted                                          ||
||     Multiple alignments : primary alignment only                           ||
|| Multi-overlapping reads : not counted                                      ||
||   Min overlapping bases : 1                                                ||
||                                                                            ||
||          Chimeric reads : not counted                                      ||
||        Both ends mapped : not required                                     ||
||                                                                            ||
\\===================== http://subread.sourceforge.net/ ======================//

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file .Rsubread_UserProvidedAnnotation_pid91858 ...         ||
||    Features : 349233                                                       ||
||    Meta-features : 60668                                                   ||
||    Chromosomes/contigs : 47                                                ||
||                                                                            ||
|| Process BAM file RNAseq_4-17-23.filtered.tagged.Aligned.out.bam...         ||
||    Paired-end reads are included.                                          ||
||    Assign alignments (paired-end) to features...                           ||
||                                                                            ||
||    WARNING: reads from the same pair were found not adjacent to each       ||
||             other in the input (due to read sorting by location or         ||
||             reporting of multi-mapping read pairs).                        ||
||                                                                            ||
||    Pairing up the read pairs.                                              ||
||                                                                            ||
||    Total alignments : 173257352                                            ||
||    Successfully assigned alignments : 70390777 (40.6%)                     ||
||    Running time : 3.70 minutes                                             ||
||                                                                            ||
||                                                                            ||
\\===================== http://subread.sourceforge.net/ ======================//

[1] "Assigning reads to features (in)"

        ==========     _____ _    _ ____  _____  ______          _____  
        =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
          =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
            ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
              ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
        ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
       Rsubread 1.32.4

//========================== featureCounts setting ===========================\\
||                                                                            ||
||             Input files : 1 BAM file                                       ||
||                           P RNAseq_4-17-23.filtered.tagged.Aligned.out ... ||
||                                                                            ||
||              Annotation : R data.frame                                     ||
||      Assignment details : <input_file>.featureCounts.bam                   ||
||                      (Note that files are saved to the output directory)   ||
||                                                                            ||
||      Dir for temp files : .                                                ||
||                 Threads : 16                                               ||
||                   Level : meta-feature level                               ||
||              Paired-end : yes                                              ||
||      Multimapping reads : counted                                          ||
||     Multiple alignments : primary alignment only                           ||
|| Multi-overlapping reads : not counted                                      ||
||   Min overlapping bases : 1                                                ||
||                                                                            ||
||          Chimeric reads : not counted                                      ||
||        Both ends mapped : not required                                     ||
||                                                                            ||
\\===================== http://subread.sourceforge.net/ ======================//

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file .Rsubread_UserProvidedAnnotation_pid91858 ...         ||
||    Features : 240846                                                       ||
||    Meta-features : 28406                                                   ||
||    Chromosomes/contigs : 34                                                ||
||                                                                            ||
|| Process BAM file RNAseq_4-17-23.filtered.tagged.Aligned.out.bam.ex.fea ... ||
||    Paired-end reads are included.                                          ||
||    Assign alignments (paired-end) to features...                           ||
||                                                                            ||
||    WARNING: reads from the same pair were found not adjacent to each       ||
||             other in the input (due to read sorting by location or         ||
||             reporting of multi-mapping read pairs).                        ||
||                                                                            ||
||    Pairing up the read pairs.                                              ||
||                                                                            ||
||    Total alignments : 173257352                                            ||
||    Successfully assigned alignments : 29420060 (17.0%)                     ||
||    Running time : 3.93 minutes                                             ||
||                                                                            ||
||                                                                            ||
\\===================== http://subread.sourceforge.net/ ======================//

[1] "2023-04-28 02:26:54 EDT"
[1] "Coordinate sorting intermediate bam file..."
[bam_sort_core] merging from 16 files and 16 in-memory blocks...
[1] "2023-04-28 02:52:43 EDT"
[1] "Hamming distance collapse in barcode chunk 1 out of 1"
[1] "Splitting data for multicore hamming distance collapse..."
[1] "Setting up multicore cluster & generating molecule mapping tables ..."
[1] "Finished multi-threaded hamming distances"
[1] "Correcting UMI barcode tags..."
Loading molecule correction dictionary...
Correcting UB tags...
[1] "6.3e+08 Reads per chunk"
[1] "2023-04-28 07:15:07 EDT"
[1] "Here are the detected subsampling options:"
[1] "Automatic downsampling"
[1] "Working on barcode chunk 1 out of 1"
[1] "Processing 2 barcodes in this chunk..."
Warning message:
In parallel::mclapply(mapList, function(tt) { :
  all scheduled cores encountered errors in user code
Error in h(simpleError(msg, call)) : 
  error in evaluating the argument 'x' in selecting a method for function 'strsplit': object 'GE' not found
Calls: convert2countM ... .makewide -> unlist -> strsplit -> .handleSimpleError -> h
Execution halted
Fri Apr 28 07:18:05 EDT 2023
Loading required package: yaml
Loading required package: Matrix
[1] "loomR found"
Error in gzfile(file, "rb") : cannot open the connection
Calls: rds_to_loom -> readRDS -> gzfile
In addition: Warning message:
In gzfile(file, "rb") :
  cannot open compressed file '/gpfs/data/lab/projects/seq/analysis/RNAseq_4-17-23/zUMIs_output/expression/RNAseq_4-17-23.dgecounts.rds', probable reason 'No such file or directory'
Execution halted
Fri Apr 28 07:18:11 EDT 2023
Descriptive statistics...
[1] "I am loading useful packages for plotting..."
[1] "2023-04-28 07:18:11 EDT"
Error in gzfile(file, "rb") : cannot open the connection
Calls: readRDS -> gzfile
In addition: Warning message:
In gzfile(file, "rb") :
  cannot open compressed file '/gpfs/data/lab/projects/seq/analysis/RNAseq_4-17-23/zUMIs_output/expression/RNAseq_4-17-23.dgecounts.rds', probable reason 'No such file or directory'
Execution halted
Fri Apr 28 07:18:26 EDT 2023

From what I can find in your code, it looks like the error is happening here:

.makewide <- function(longdf,type){
  dt<-longdf[, list(GEu=unlist(strsplit(GE,","))), by=c("RG","GE",type)][ #this splits multioverlap gene lists by comma
             , cnt := get(type) / (stringr::str_count(GE,",")+1) ][
             , list(tot=sum(cnt)),by=c("RG","GEu")  ]

Though I don't understand how zUMIs works, it seems that GE is a tag loaded from one of the BAM files? However, looking through all the BAM files made by zUMIs, I don't see a GE tag anywhere.

What is causing this bug?

Thanks

Sorry, correction: the .filtered.Aligned.GeneTagged.UBcorrected.sorted.bam file DOES have GE tags. So I'm not sure why this bug is happening. The GE tag should be retrieved and therefore the GE objects should exist when that strsplit(GE,",") function runs.

I think the same error came up recently in another issue, which R version do you use? It was solved by using the conda environment you get with zUMIs, please try that! zUMIs.sh -c -y config.yaml

R 4.2.2

I saw in another issue on here that R 3.6 fixes this? Is that correct?

I tried '-c' option but that gives even more errors. Conda is not completely isolated from the local environment so I think it still has issues.

Any other suggestions? I think maybe the only other option is docker.

But it would be great to understand why this 'GE' error is happening in the first place. That would fix the issue.

Sorry one more idea: is it possible to run without '-c' and then rerun with '-c' and it will continue from the point that the initial run failed at? That could be a solution. Would the rerun with '-c' detect the prior output files and skip to the last step that has not been completed? That way I can get to the 'GE' error step without '-c' and then rerun with '-c' to complete the pipline.

It looks like the conda environment of zUMIs does not have R installed. So a docker where zUMIs is cloned into does not have R. I'd suggest putting R in the conda environment or making a fully containerized docker with all requirements.

Which R version should I install?

Thanks

Hi,

The conda environment has R preinstalled. If you think it doesn't in your copy, try to clone again from GitHub.

Here is also a full conda export for your reference.

channels:
  - davemcg
  - ccb-sb
  - conda-forge
  - bioconda
  - defaults
dependencies:
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  - r-highr=0.9=r36hc72bb7e_0
  - r-hms=1.1.0=r36hc72bb7e_0
  - r-htmltools=0.5.1.1=r36h03ef668_0
  - r-htmlwidgets=1.5.3=r36hc72bb7e_0
  - r-httpuv=1.6.1=r36h03ef668_0
  - r-httr=1.4.2=r36h6115d3f_0
  - r-inflection=1.3.5=r36h6115d3f_0
  - r-ini=0.3.1=r36h6115d3f_1003
  - r-isoband=0.2.4=r36h03ef668_0
  - r-iterators=1.0.13=r36h142f84f_0
  - r-itertools=0.1_3=r36_1003
  - r-jquerylib=0.1.4=r36hc72bb7e_0
  - r-jsonlite=1.7.2=r36hcfec24a_0
  - r-knitr=1.33=r36hc72bb7e_0
  - r-labeling=0.4.2=r36h142f84f_0
  - r-lambda.r=1.2.4=r36h6115d3f_1
  - r-later=1.2.0=r36h03ef668_0
  - r-lattice=0.20_44=r36hcfec24a_0
  - r-lazyeval=0.2.2=r36hcfec24a_2
  - r-lifecycle=1.0.0=r36hc72bb7e_0
  - r-loomr=0.2.0=r36_0
  - r-magrittr=2.0.1=r36hcfec24a_1
  - r-markdown=1.1=r36hcfec24a_1
  - r-mass=7.3_54=r36hcfec24a_0
  - r-matrix=1.3_3=r36he454529_0
  - r-matrixstats=0.58.0=r36hcfec24a_0
  - r-mclust=5.4.7=r36h52d45c5_0
  - r-memoise=2.0.0=r36hc72bb7e_0
  - r-mgcv=1.8_35=r36he454529_0
  - r-mime=0.10=r36hcfec24a_0
  - r-munsell=0.5.0=r36h6115d3f_1003
  - r-nlme=3.1_152=r36h859d828_0
  - r-openssl=1.4.4=r36he36bf35_0
  - r-pillar=1.6.1=r36hc72bb7e_0
  - r-pkgbuild=1.2.0=r36hc72bb7e_0
  - r-pkgconfig=2.0.3=r36h6115d3f_1
  - r-pkgload=1.2.1=r36h03ef668_0
  - r-plogr=0.2.0=r36h6115d3f_1003
  - r-praise=1.0.0=r36h6115d3f_1004
  - r-prettyunits=1.1.1=r36h6115d3f_1
  - r-processx=3.5.2=r36hcfec24a_0
  - r-progress=1.2.2=r36h6115d3f_2
  - r-promises=1.2.0.1=r36h03ef668_0
  - r-ps=1.6.0=r36hcfec24a_0
  - r-purrr=0.3.4=r36hcfec24a_1
  - r-r6=2.5.0=r36hc72bb7e_0
  - r-rappdirs=0.3.3=r36hcfec24a_0
  - r-rcmdcheck=1.3.3=r36h6115d3f_3
  - r-rcolorbrewer=1.1_2=r36h6115d3f_1003
  - r-rcpp=1.0.6=r36h03ef668_0
  - r-rcurl=1.98_1.3=r36hcfec24a_0
  - r-rematch2=2.1.2=r36h6115d3f_1
  - r-remotes=2.3.0=r36hc72bb7e_0
  - r-rex=1.2.0=r36h6115d3f_1
  - r-rlang=0.4.11=r36hcfec24a_0
  - r-rlist=0.4.6.1=r36h6115d3f_1003
  - r-roxygen2=7.1.1=r36h0357c0b_0
  - r-rprojroot=2.0.2=r36hc72bb7e_0
  - r-rsqlite=2.2.5=r36h03ef668_0
  - r-rstudioapi=0.13=r36hc72bb7e_0
  - r-rversions=2.0.2=r36h6115d3f_0
  - r-sass=0.4.0=r36h03ef668_0
  - r-scales=1.1.1=r36h6115d3f_0
  - r-sessioninfo=1.1.1=r36h6115d3f_1002
  - r-shiny=1.6.0=r36hc72bb7e_0
  - r-shinybs=0.61=r36h6115d3f_1003
  - r-shinythemes=1.2.0=r36hc72bb7e_0
  - r-snow=0.4_3=r36h6115d3f_1002
  - r-sourcetools=0.1.7=r36he1b5a44_1002
  - r-stringdist=0.9.6.3=r36hcfec24a_0
  - r-stringi=1.6.2=r36hcabe038_0
  - r-stringr=1.4.0=r36h6115d3f_2
  - r-sys=3.4=r36hcfec24a_0
  - r-testthat=3.0.2=r36h03ef668_0
  - r-tibble=3.1.2=r36hcfec24a_0
  - r-tidyselect=1.1.1=r36hc72bb7e_0
  - r-usethis=2.0.1=r36hc72bb7e_0
  - r-utf8=1.2.1=r36hcfec24a_0
  - r-vctrs=0.3.8=r36hcfec24a_1
  - r-viridislite=0.4.0=r36hc72bb7e_0
  - r-waldo=0.2.5=r36hc72bb7e_0
  - r-whisker=0.4=r36h6115d3f_1
  - r-withr=2.4.2=r36hc72bb7e_0
  - r-xfun=0.23=r36hcfec24a_0
  - r-xml=3.99_0.3=r36hcdcec82_1
  - r-xml2=1.3.2=r36h0357c0b_1
  - r-xopen=1.0.0=r36h6115d3f_1003
  - r-xtable=1.8_4=r36h6115d3f_3
  - r-yaml=2.2.1=r36hcfec24a_1
  - r-zip=2.1.1=r36hcfec24a_0
  - readline=8.1=h46c0cb4_0
  - samtools=1.13=h8c37831_0
  - scikit-learn=0.24.2=py39h4dfa638_1
  - scipy=1.7.1=py39hee8e79c_0
  - sed=4.8=he412f7d_0
  - setuptools=57.4.0=py39hf3d152e_0
  - six=1.16.0=pyh6c4a22f_0
  - sqlite=3.36.0=h9cd32fc_0
  - star=2.7.3a=0
  - sysroot_linux-64=2.12=he073ed8_14
  - threadpoolctl=2.2.0=pyh8a188c0_0
  - tk=8.6.11=h21135ba_0
  - tktable=2.10=hb7b940f_3
  - tornado=6.1=py39h3811e60_1
  - tzdata=2021a=he74cb21_1
  - velocyto.py=0.17.17=py39hcbe4a3b_4
  - wheel=0.37.0=pyhd8ed1ab_1
  - xorg-kbproto=1.0.7=h7f98852_1002
  - xorg-libice=1.0.10=h7f98852_0
  - xorg-libsm=1.2.3=hd9c2040_1000
  - xorg-libx11=1.7.2=h7f98852_0
  - xorg-libxau=1.0.9=h7f98852_0
  - xorg-libxdmcp=1.1.3=h7f98852_0
  - xorg-libxext=1.3.4=h7f98852_1
  - xorg-libxrender=0.9.10=h7f98852_1003
  - xorg-libxt=1.2.1=h7f98852_2
  - xorg-renderproto=0.11.1=h7f98852_1002
  - xorg-xextproto=7.3.0=h7f98852_1002
  - xorg-xproto=7.0.31=h7f98852_1007
  - xz=5.2.5=h516909a_1
  - zlib=1.2.11=h516909a_1010
  - zstd=1.5.0=ha95c52a_0

I have confirmed running in base system docker and AWS cloud instances previously.

Best, Christoph

Thanks. I think the issue was #332 . I removed zUMIs-env and now it is working. Now I'll run the pipeline again from docker and hopefully it works.

Hi @gevro , I met the same problem "function 'strsplit': object 'GE' not found". I used my own zUMIs environment with R version 4.3.1. Could you please tell me have you solved this problem?

I wasn't able to figure this out, sorry.

I see. But wandering did you still use it? I mean how you treat this problem finally?

sdparekh / zUMIs

'strsplit': object 'GE' not foundBUG #358