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singleron-RD / scrna

Pipeline for Single-cell RNA Seq
MIT License
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Please fix, why the required file is not generated? Please share the proper config file for single cell RNA #5

Open tiancaigg opened 2 months ago

tiancaigg commented 2 months ago

Description of the bug

Missing output file(s) *.Solo.out/GeneFull_Ex50pAS/CellReads.stats expected by process SINGLERONRD_SCRNA:SCRNA:STARSOLO (JW121_E) Please fix, why the required file is not generated? Please give me proper config file for single cell RNA!!!!

Command used and terminal output

/media/HDD2/donghui/Singleron » nextflow run singleron-RD/scrna \ 1 ↵ hu@chaelab2 --input ./samplesheet-5per.csv \ --outdir ./results_singularity_5percent_1 \ --star_genome /media/HDD2/Genomes/Ath_Ensembl56_modified_pJW121/Ath_STAR_index_pJW121 \ -profile singularity \ --max_cpus 100 \ --max_memory '190.GB' \ -qs 10 \ -c ./config_v4.config

N E X T F L O W ~ version 24.04.4

WARNING: Could not load nf-core/config profiles: null/nfcore_custom.config Launching https://github.com/singleron-RD/scrna [focused_church] 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 : focused_church containerEngine : singularity launchDir : /media/HDD2/donghui/Singleron workDir : /media/HDD2/donghui/Singleron/work projectDir : /home/hu/.nextflow/assets/singleron-RD/scrna userName : hu profile : singularity configFiles :

Input/output options input : ./samplesheet-5per.csv outdir : ./results_singularity_5percent_1

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

Max job request options max_cpus : 100 max_memory : 190.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:

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/sub2_0.05.fq.gz 1/sub1_0.05.fq.gz \ --genomeDir Ath_STAR_index_pJW121 \ --outFileNamePrefix JW121_E. \ --runThreadN 110 \ --limitBAMsortRAM 100091904630 --outBAMsortingBinsN 50 --outBAMsortingThreadN 30 --soloFeatures GeneFull_Ex50pAS --outSAMtype BAM SortedByCoordinate --outSAMattributes NH HI AS nM CR UR CB UB GX GN

cat <<-END_VERSIONS > versions.yml "SINGLERONRDSCRNA:SCRNA:STARSOLO": star: $(STAR --version | sed -e "s/STAR//g") END_VERSIONS Command exit status: 0

Command output: Sep 05/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/sub2_0.05.fq.gz 1/sub1_0.05.fq.gz --genomeDir Ath_STAR_index_pJW121 --outFileNamePrefix JW121_E. --runThreadN 110 --limitBAMsortRAM 100091904630 --outBAMsortingBinsN 50 --outBAMsortingThreadN 30 --soloFeatures GeneFull_Ex50pAS --outSAMtype BAM SortedByCoordinate --outSAMattributes NH HI AS nM CR UR CB UB GX GN Sep 05STAR version: 2.7.11b compiled: 2024-01-29T15:15:38+0000 :/opt/conda/conda-bld/star_1706541070242/work/source Sep 05 12:38:36 ..... started STAR run Sep 05 12:38:36 ..... loading genome Sep 05 12:38:38 ..... started mapping Sep 05 12:44:20 ..... finished mapping Sep 05 12:44:21 ..... started Solo counting Sep 05 12:44:40 ..... finished Solo counting Sep 05 12:44:40 ..... started sorting BAM Sep 05 12:45:15 ..... finished successfully

Work dir: /media/HDD2/donghui/Singleron/work/ff/3bb2ed5f148752ea7730aa635c4079

Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named .command.sh

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

Relevant files

config file """ process { withName: 'SINGLERONRD_SCRNA:SCRNA:STARSOLO' { cpus = 110 memory = '200 GB' ext.args = "--limitBAMsortRAM 100091904630 --outBAMsortingBinsN 50 --outBAMsortingThreadN 30 --soloFeatures GeneFull_Ex50pAS --outSAMtype BAM SortedByCoordinate --outSAMattributes NH HI AS nM CR UR CB UB GX GN" } }

params { max_cpus = 88 }"""

System information

No response

zhouyiqi91 commented 2 months ago

I think it is because you have overwritten the ext.args of starsolo with the "-c" argument. Without "--soloCellReadStats Standard", CellReadStats will not be generated. https://github.com/singleron-RD/scrna/blob/master/conf/modules.config#L37

From the nextflow document:

Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters;

If you want to change the paramters of starsolo, you can change the provided paramters here: https://github.com/singleron-RD/scrna/blob/master/docs/parameters.md#starsolo-options

tiancaigg commented 2 months ago

I think it is because you have overwritten the ext.args of starsolo with the "-c" argument. Without "--soloCellReadStats Standard", CellReadStats will not be generated. https://github.com/singleron-RD/scrna/blob/master/conf/modules.config#L37

From the nextflow document:

Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters;

If you want to change the paramters of starsolo, you can change the provided paramters here: https://github.com/singleron-RD/scrna/blob/master/docs/parameters.md#starsolo-options

the reason I use custom config is because STAR-avx2 only run 6 thread in my 128 thread 256 GB mem computer. I need to wait days for it to complete. how to increase thread without using custom config?

zhouyiqi91 commented 2 months ago

By default, starsolo uses max 12 cpus. https://github.com/singleron-RD/scrna/blob/master/conf/base.config#L43

You can tweek this value by specify process resources in the config file.

my.config

process {
    withLabel:process_high {
        cpus   = {24}
        memory = {32.GB}
        time   = {48.h}
    }
}

then

nextflow run ... -c my.config

BTW, I remember the developer of STAR once said that using more than 20 cpu would do little help.

tiancaigg commented 2 months ago

-c my.config will override any other config, then I need all the paramters in my.config

zhouyiqi91 commented 2 months ago

I think all the pipeline parameters, like "--input", "--fasta" should use CLI or params file.

This should work

nextflow run singleron-RD/scrna \ 
--input ./samplesheet-5per.csv \
--outdir ./results_singularity_5percent_1 \
--star_genome /media/HDD2/Genomes/Ath_Ensembl56_modified_pJW121/Ath_STAR_index_pJW121 \
-profile singularity \
-c my.config \
--max_cpus 100
zhouyiqi91 commented 2 months ago

I have updated a new version 1.2.2 with argument --star_cpus

nextflow run singleron-RD/scrna ... -r 1.2.2  --star_cpus 24