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singleron-RD / scrna

Pipeline for Single-cell RNA Seq
MIT License
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--limitBAMsortRAM #6

Open tiancaigg opened 1 month ago

tiancaigg commented 1 month ago

Description of the bug

--limitBAMsortRAM cannot be set by --star_options '--limitBAMsortRAM 800000000000', it can only be set by custom config, which override any other existing config.

I've already set export NXF_OPTS='-Xms1g -Xmx180g' in .zshrc

Command used and terminal output

/media/HDD2/donghui/Singleron »                                                1sleep 1hchaelab2
nextflow run singleron-RD/scrna \     2h
--input ./samplesheet.csv \
--outdir ./results_no_custom_config \
--star_genome /media/HDD2/Genomes/Ath_Ensembl56_modified_pJW121/Ath_STAR_index_pJW121 \
-profile docker \
--max_cpus 50 \
--max_memory '100.GB' \
-qs 10

 N E X T F L O W   ~  version 24.04.4

NOTE: Your local project version looks outdated - a different revision is available in the remote repository [8e43e39fce]
WARNING: Could not load nf-core/config profiles: null/nfcore_custom.config
Launching `https://github.com/singleron-RD/scrna` [compassionate_solvay] DSL2 - revision: 07f6143fe8 [master]

WARN: Access to undefined parameter `monochromeLogs` -- Initialise it to a default value eg. `params.monochromeLogs = some_value`

------------------------------------------------------
                                        ,--./,-.
        ___     __   __   __   ___     /,-._.--~'
  |\ | |__  __ /  ` /  \ |__) |__         }  {
  | \| |       \__, \__/ |  \ |___     \`-._,-`-,
                                        `._,._,'
  singleron-RD/scrna v1.2.1-g07f6143
------------------------------------------------------
Core Nextflow options
  revision        : master
  runName         : compassionate_solvay
  containerEngine : docker
  launchDir       : /media/HDD2/donghui/Singleron
  workDir         : /media/HDD2/donghui/Singleron/work
  projectDir      : /home/hu/.nextflow/assets/singleron-RD/scrna
  userName        : hu
  profile         : docker
  configFiles     : 

Input/output options
  input           : ./samplesheet.csv
  outdir          : ./results_no_custom_config

Genome
  star_genome     : /media/HDD2/Genomes/Ath_Ensembl56_modified_pJW121/Ath_STAR_index_pJW121

Max job request options
  max_cpus        : 50
  max_memory      : 100.GB

Generic options
  publish_dir_mode: symlink

!! Only displaying parameters that differ from the pipeline defaults !!
------------------------------------------------------
If you use singleron-RD/scrna for your analysis please cite:

* The pipeline

* The nf-core framework
  https://doi.org/10.1038/s41587-020-0439-x

* Software dependencies
  https://github.com/singleron-RD/scrna/blob/master/CITATIONS.md
------------------------------------------------------
executor >  local (2)
[8d/869bd9] SINGLERONRD_SCRNA:SCRNA:PROTOCOL_CMD (JW121_E) [100%] 1 of 1 ✔
[be/a9fbd6] SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)     [  0%] 0 of 1
[-        ] SINGLERONRD_SCRNA:SCRNA:CELL_CALLING           -
[-        ] SINGLERONRD_SCRNA:SCRNA:STARSOLO_SUMMARY       -
[-        ] SINGLERONRD_SCRNA:SCRNA:MULTIQC                -
ERROR ~ Error executing process > 'SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)'

Caused by:
  Process `SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)` terminated with an error exit status (102)

Command executed:

  STAR \
      --soloType CB_UMI_Complex  --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2  --soloUMIposition 0_60_0_71 --soloUMIlen 12  --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt  --readFilesCommand zcat \
      --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz \
      --genomeDir Ath_STAR_index_pJW121 \
      --outFileNamePrefix JW121_E. \
      --runThreadN 12 \
      --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordinate

  cat <<-END_VERSIONS > versions.yml
  "SINGLERONRD_SCRNA:SCRNA:STARSOLO":
      star: $(STAR --version | sed -e "s/STAR_//g")
  END_VERSIONS

Command exit status:
  102

Command output:
        /usr/local/bin/STAR-avx2 --soloType CB_UMI_Complex --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2 --soloUMIposition 0_60_0_71 --soloUMIlen 12 --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt --readFilesCommand zcat --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz --genomeDir Ath_STAR_index_pJW121 --outFileNamePrefix JW121_E. --runThreadN 12 --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordinate
        STAR version: 2.7.11b   compiled: 2024-01-29T15:15:38+0000 :/opt/conda/conda-bld/star_1706541070242/work/source
  Sep 05 13:06:39 ..... started STAR run
  Sep 05 13:06:39 ..... loading genome
  Sep 05 13:06:55 ..... started mapping
  Sep 05 20:55:27 ..... finished mapping
  Sep 05 20:55:27 ..... started Solo counting
  Sep 05 21:03:50 ..... finished Solo counting
  Sep 05 21:03:50 ..... started sorting BAM

Command error:
        /usr/local/bin/STAR-avx2 --soloType CB_UMI_Complex --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2 --soloUMIposition 0_60_0_71 --soloUMIlen 12 --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt --readFilesCommand zcat --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz --genomeDir Ath_STAR_index_pJW121 --outFileNamePrefix JW121_E. --runThreadN 12 --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordiexecutor >  local (2)
[8d/869bd9] SINGLERONRD_SCRNA:SCRNA:PROTOCOL_CMD (JW121_E) [100%] 1 of 1 ✔      [be/a9fbd6] SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)     [100%] 1 of 1, failed: 1 ✘
[-        ] SINGLERONRD_SCRNA:SCRNA:CELL_CALLING           -
[-        ] SINGLERONRD_SCRNA:SCRNA:STARSOLO_SUMMARY       -
[-        ] SINGLERONRD_SCRNA:SCRNA:MULTIQC                -
Execution cancelled -- Finishing pending tasks before exit
-[singleron-RD/scrna] Pipeline completed with errors-
ERROR ~ Error executing process > 'SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)'

Caused by:
  Process `SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)` terminated with an error exit status (102)

Command executed:

  STAR \                                                                              --soloType CB_UMI_Complex  --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2  --soloUMIposition 0_60_0_71 --soloUMIlen 12  --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt  --readFilesCommand zcat \
      --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz \
      --genomeDir Ath_STAR_index_pJW121 \
      --outFileNamePrefix JW121_E. \
      --runThreadN 12 \
      --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordinate

  cat <<-END_VERSIONS > versions.yml
  "SINGLERONRD_SCRNA:SCRNA:STARSOLO":
      star: $(STAR --version | sed -e "s/STAR_//g")
  END_VERSIONS

Command exit status:
  102

Command output:
        /usr/local/bin/STAR-avx2 --soloType CB_UMI_Complex --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2 --soloUMIposition 0_60_0_71 --soloUMIlen 12 --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt --readFilesCommand zcat --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz --genomeDir Ath_STAR_index_pJW121 --outFileNamePrefix JW121_E. --runThreadN 12 --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordinate
        STAR version: 2.7.11b   compiled: 2024-01-29T15:15:38+0000 :/opt/conda/conda-bld/star_1706541070242/work/source
  Sep 05 13:06:39 ..... started STAR run
  Sep 05 13:06:39 ..... loading genome
  Sep 05 13:06:55 ..... started mapping
  Sep 05 20:55:27 ..... finished mapping
  Sep 05 20:55:27 ..... started Solo counting
  Sep 05 21:03:50 ..... finished Solo counting
  Sep 05 21:03:50 ..... started sorting BAM

Command error:
        /usr/local/bin/STAR-avx2 --soloType CB_UMI_Complex --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2 --soloUMIposition 0_60_0_71 --soloUMIlen 12 --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt --readFilesCommand zcat --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz --genomeDir Ath_STAR_index_pJW121 --outFileNamePrefix JW121_E. --runThreadN 12 --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordiexecutor >  local (2)
[8d/869bd9] SINGLERONRD_SCRNA:SCRNA:PROTOCOL_CMD (JW121_E) [100%] 1 of 1 ✔      [be/a9fbd6] SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)     [100%] 1 of 1, failed: 1 ✘
[-        ] SINGLERONRD_SCRNA:SCRNA:CELL_CALLING           -
[-        ] SINGLERONRD_SCRNA:SCRNA:STARSOLO_SUMMARY       -
[-        ] SINGLERONRD_SCRNA:SCRNA:MULTIQC                -
Execution cancelled -- Finishing pending tasks before exit
-[singleron-RD/scrna] Pipeline completed with errors-
ERROR ~ Error executing process > 'SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)'

Caused by:
  Process `SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E)` terminated with an error exit status (102)

Command executed:

  STAR \                                                                              --soloType CB_UMI_Complex  --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2  --soloUMIposition 0_60_0_71 --soloUMIlen 12  --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt  --readFilesCommand zcat \
      --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz \
      --genomeDir Ath_STAR_index_pJW121 \
      --outFileNamePrefix JW121_E. \
      --runThreadN 12 \
      --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordinate

  cat <<-END_VERSIONS > versions.yml
  "SINGLERONRD_SCRNA:SCRNA:STARSOLO":
      star: $(STAR --version | sed -e "s/STAR_//g")
  END_VERSIONS

Command exit status:
  102

Command output:
        /usr/local/bin/STAR-avx2 --soloType CB_UMI_Complex --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2 --soloUMIposition 0_60_0_71 --soloUMIlen 12 --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt --readFilesCommand zcat --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz --genomeDir Ath_STAR_index_pJW121 --outFileNamePrefix JW121_E. --runThreadN 12 --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordinate
        STAR version: 2.7.11b   compiled: 2024-01-29T15:15:38+0000 :/opt/conda/conda-bld/star_1706541070242/work/source
  Sep 05 13:06:39 ..... started STAR run
  Sep 05 13:06:39 ..... loading genome
  Sep 05 13:06:55 ..... started mapping
  Sep 05 20:55:27 ..... finished mapping
  Sep 05 20:55:27 ..... started Solo counting
  Sep 05 21:03:50 ..... finished Solo counting
  Sep 05 21:03:50 ..... started sorting BAM

Command error:
        /usr/local/bin/STAR-avx2 --soloType CB_UMI_Complex --soloCBposition 0_0_0_8 0_25_0_33 0_50_0_58 --soloCBmatchWLtype EditDist_2 --soloUMIposition 0_60_0_71 --soloUMIlen 12 --soloCBwhitelist assets/whitelist/GEXSCOPE-V2/bc1.txt assets/whitelist/GEXSCOPE-V2/bc2.txt assets/whitelist/GEXSCOPE-V2/bc3.txt --readFilesCommand zcat --readFilesIn 2/DH1_2_R-R2.fq.gz 1/DH1_2_R-R1.fq.gz --genomeDir Ath_STAR_index_pJW121 --outFileNamePrefix JW121_E. --runThreadN 12 --soloFeatures GeneFull_Ex50pAS --outFilterMatchNmin 50 --outSAMattributes NH HI nM AS CR UR CB UB GX GN sF --soloCellFilter CellRanger2.2 --soloCellReadStats Standard --soloBarcodeReadLength 0 --clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordinate
        STAR version: 2.7.11b   compiled: 2024-01-29T15:15:38+0000 :/opt/conda/conda-bld/star_1706541070242/work/source
  Sep 05 13:06:39 ..... started STAR run
  Sep 05 13:06:39 ..... loading genome
  Sep 05 13:06:55 ..... started mapping
  Sep 05 20:55:27 ..... finished mapping
  Sep 05 20:55:27 ..... started Solo counting

  EXITING because of fatal ERROR: not enough memory for BAM sorting: 
  SOLUTION: re-run STAR with at least --limitBAMsortRAM 5051923277
  Sep 05 21:03:50 ...... FATAL ERROR, exiting
  Sep 05 21:03:50 ..... finished Solo counting
  Sep 05 21:03:50 ..... started sorting BAM

Work dir:
  /media/HDD2/donghui/Singleron/work/be/a9fbd60ff2f4b0195f37dbb3719da8

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line

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

Relevant files

No response

System information

No response

tiancaigg commented 1 month ago

when publish new version, please run a real dataset, don't just run toy dataset

zhouyiqi91 commented 1 month ago

We have tested various samples internally and have not encountered similar problems. I am not sure why this problem occurs, but I guess it is related to the genome and amount of data used. https://github.com/alexdobin/STAR/issues/870

Anyway, you can always add new starsolo parameters through starsolo_extra_args:

--starsolo_extra_args "--clip3pAdapterSeq AAAAAAAAAAAA --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 10000000000"

Also, the memory limit in NXF_OPTS should not be set too high, generally 4GB is enough. https://github.com/nextflow-io/nextflow/issues/1567 https://github.com/singleron-RD/scrna/blob/master/docs/usage.md#nextflow-memory-requirements