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Limitted input length. #86

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Fabian-Boehm opened this issue Nov 23, 2023 · 2 comments
Closed

Limitted input length. #86

Fabian-Boehm opened this issue Nov 23, 2023 · 2 comments
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@nictru
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@Fabian-Boehm
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Fabian-Boehm commented Nov 23, 2023 •
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###Description of the bug

From what I understood from the error messages the maximum input length of this pipeline is at 650 bases per read.

For reads longer 650 the RNA sequence seems to be cropped to a length of 650, while the quality string doesn't, leading to inequal quality string and sequence length.

Command used and terminal output

nextflow run /nfs/data3/CIRCEST/pipeline -profile apptainer,cluster -params-file ./configs/params.yaml

PARAMS:
input: './samplesheet.csv'
outdir: './results/'
save_reference: true
save_intermediates:  true
hisat2_build_memory:  '200.GB'
genome:  'WBcel235'
star: null
module: 'circrna_discovery'


ERROR ~ Error executing process > 'NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_1ST_PASS (elegans_unselected_1)'

Caused by:
  Process `NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_1ST_PASS (elegans_unselected_1)` terminated with an error exit status (104)

Command executed:

  STAR \
      --genomeDir star \
      --readFilesIn input1/elegans_unselected_1_trimmed.fq.gz  \
      --runThreadN 24 \
      --outFileNamePrefix elegans_unselected_1. \
      --outSAMtype BAM Unsorted \
       \
      --outSAMattrRGline 'ID:elegans_unselected_1'  'SM:elegans_unselected_1'  \
      --chimOutType Junctions WithinBAM --outSAMunmapped Within --outFilterType BySJout --outReadsUnmapped None --readFilesCommand zcat --alignSJDBoverhangMin 10 --chimJunctionOverhangMin 10 --chimSegmentMin 10



  if [ -f elegans_unselected_1.Unmapped.out.mate1 ]; then
      mv elegans_unselected_1.Unmapped.out.mate1 elegans_unselected_1.unmapped_1.fastq
      gzip elegans_unselected_1.unmapped_1.fastq
  fi
  if [ -f elegans_unselected_1.Unmapped.out.mate2 ]; then
      mv elegans_unselected_1.Unmapped.out.mate2 elegans_unselected_1.unmapped_2.fastq
      gzip elegans_unselected_1.unmapped_2.fastq
  fi

  cat <<-END_VERSIONS > versions.yml
  "NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_1ST_PASS":
executor >  slurm (7)
[54/b6824f] process > NFCORE_CIRCRNA:CIRCRNA:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet.csv)                                     [100%] 1 of 1 ✔
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CAT_FASTQ                                                                           -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:PREPARE_GENOME:BOWTIE_BUILD                                                         -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:PREPARE_GENOME:BOWTIE2_BUILD                                                        -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:PREPARE_GENOME:BWA_INDEX                                                            -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:PREPARE_GENOME:HISAT2_EXTRACTSPLICESITES                                            -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:PREPARE_GENOME:HISAT2_BUILD                                                         -
[5b/f8b873] process > NFCORE_CIRCRNA:CIRCRNA:PREPARE_GENOME:STAR_GENOMEGENERATE (genome.fa)                                      [100%] 1 of 1 ✔
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:PREPARE_GENOME:SEGEMEHL_INDEX                                                       -
[00/9b89ee] process > NFCORE_CIRCRNA:CIRCRNA:FASTQC_TRIMGALORE:FASTQC (elegans_unselected_1)                                     [100%] 1 of 1 ✔
[95/3c54c8] process > NFCORE_CIRCRNA:CIRCRNA:FASTQC_TRIMGALORE:TRIMGALORE (elegans_unselected_1)                                 [100%] 1 of 1 ✔
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:SEGEMEHL_ALIGN                                                    -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:SEGEMEHL_FILTER                                                   -
[17/6ccdf3] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_1ST_PASS (elegans_unselected_1)                              [100%] 2 of 2, failed: 2..
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_SJDB                                                         -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_2ND_PASS                                                     -
[39/0919bd] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CIRCEXPLORER2_REF (genes.gtf)                                     [100%] 1 of 1 ✔
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CIRCEXPLORER2_PAR                                                 -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CIRCEXPLORER2_ANN                                                 -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CIRCEXPLORER2_FLT                                                 -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CIRCRNA_FINDER_FILTER                                             -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:FIND_CIRC_ALIGN                                                   -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:SAMTOOLS_INDEX                                                    -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:SAMTOOLS_VIEW                                                     -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:FIND_CIRC_ANCHORS                                                 -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:FIND_CIRC                                                         -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:FIND_CIRC_FILTER                                                  -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CIRIQUANT_YML                                                     -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CIRIQUANT                                                         -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CIRIQUANT_FILTER                                                  -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:DCC_MATE1_1ST_PASS                                                -                          [-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:DCC_MATE1_SJDB                                                    -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:DCC_MATE1_2ND_PASS                                                -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:DCC_MATE2_1ST_PASS                                                -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:DCC_MATE2_SJDB                                                    -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:DCC_MATE2_2ND_PASS                                                -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:DCC                                                               -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:DCC_FILTER                                                        -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:MAPSPLICE_REFERENCE                                               -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:MAPSPLICE_ALIGN                                                   -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:MAPSPLICE_PARSE                                                   -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:MAPSPLICE_ANNOTATE                                                -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:MAPSPLICE_FILTER                                                  -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:COUNTS_SINGLE                                                     -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:REMOVE_HEADER                                                     -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:SPLIT_ANNOTATION                                                  -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:ANNOTATION                                                        -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:CAT_ANNOTATION                                                    -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:SORT_ANNOTATION                                                   -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:FASTA                                                             -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:PSIRC_INDEX                                                       -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:PSIRC_QUANT                                                       -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:PSIRC_COMBINE                                                     -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:MIRNA_PREDICTION:TARGETSCAN_DATABASE                                                -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:MIRNA_PREDICTION:TARGETSCAN                                                         -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:MIRNA_PREDICTION:MIRANDA                                                            -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:MIRNA_PREDICTION:MIRNA_TARGETS                                                      -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:HISAT2_ALIGN                                                -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:BAM_SORT_STATS_SAMTOOLS:SAMTOOLS_SORT                       -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:BAM_SORT_STATS_SAMTOOLS:SAMTOOLS_INDEX                      -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:BAM_SORT_STATS_SAMTOOLS:BAM_STATS_SAMTOOLS:SAMTOOLS_STATS   -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:BAM_SORT_STATS_SAMTOOLS:BAM_STATS_SAMTOOLS:SAMTOOLS_FLAG... -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:BAM_SORT_STATS_SAMTOOLS:BAM_STATS_SAMTOOLS:SAMTOOLS_IDXS... -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:STRINGTIE_STRINGTIE                                         -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:STRINGTIE_PREPDE                                            -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:DESEQ2_DIFFERENTIAL_EXPRESSION                              -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:PARENT_GENE                                                 -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:PREPARE_CLR_TEST                                            -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:DIFFERENTIAL_EXPRESSION:CIRCTEST                                                    -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:CUSTOM_DUMPSOFTWAREVERSIONS                                                         -
[-        ] process > NFCORE_CIRCRNA:CIRCRNA:MULTIQC                                                                             -
ERROR ~ Error executing process > 'NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_1ST_PASS (elegans_unselected_1)'

Caused by:
  Process `NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_1ST_PASS (elegans_unselected_1)` terminated with an error exit status (104)

Command executed:

  STAR \
      --genomeDir star \
      --readFilesIn input1/elegans_unselected_1_trimmed.fq.gz  \
      --runThreadN 24 \
      --outFileNamePrefix elegans_unselected_1. \
      --outSAMtype BAM Unsorted \
       \
      --outSAMattrRGline 'ID:elegans_unselected_1'  'SM:elegans_unselected_1'  \
      --chimOutType Junctions WithinBAM --outSAMunmapped Within --outFilterType BySJout --outReadsUnmapped None --readFilesCommand zcat --alignSJDBoverhangMin 10 --chimJunctionOverhangMin 10 --chimSegmentMin 10



  if [ -f elegans_unselected_1.Unmapped.out.mate1 ]; then
      mv elegans_unselected_1.Unmapped.out.mate1 elegans_unselected_1.unmapped_1.fastq
      gzip elegans_unselected_1.unmapped_1.fastq
  fi
  if [ -f elegans_unselected_1.Unmapped.out.mate2 ]; then
      mv elegans_unselected_1.Unmapped.out.mate2 elegans_unselected_1.unmapped_2.fastq
      gzip elegans_unselected_1.unmapped_2.fastq
  fi

  cat <<-END_VERSIONS > versions.yml
  "NFCORE_CIRCRNA:CIRCRNA:CIRCRNA_DISCOVERY:STAR_1ST_PASS":
      star: $(STAR --version | sed -e "s/STAR_//g")
      samtools: $(echo $(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*$//')
      gawk: $(echo $(gawk --version 2>&1) | sed 's/^.*GNU Awk //; s/, .*$//')
  END_VERSIONS

Command exit status:
  104

Command output:
        STAR --genomeDir star --readFilesIn input1/elegans_unselected_1_trimmed.fq.gz --runThreadN 24 --outFileNamePrefix elegans_unselected_1. --outSAMtype BAM Unsorted --outSAMattrRGline ID:elegans_unselected_1 SM:elegans_unselected_1 --chimOutType Junctions WithinBAM --outSAMunmapped Within --outFilterType BySJout --outReadsUnmapped None --readFilesCommand zcat --alignSJDBoverhangMin 10 --chimJunctionOverhangMin 10 --chimSegmentMin 10
        STAR version: 2.7.9a   compiled: 2021-05-04T09:43:56-0400 vega:/home/dobin/data/STAR/STARcode/STAR.master/source
  Nov 23 14:00:32 ..... started STAR run
  Nov 23 14:00:32 ..... loading genome
  Nov 23 14:00:34 ..... started mapping

Command error:

  EXITING because of FATAL ERROR in reads input: quality string length is not equal to sequence length
  @SRR19055922.2.1
  AGAATTGGCTCTAGAGAATGCAGATATCATTGAGGTCGAGACCAAAAAGCCTTACAAGACTAAAGAATAAGAAAAACTGTTTTCACAGCAATAACAGAATTGAAAAGATCCATGATTACGCACCTCACTGGTCTCGAGAATGTCATGCAAGAGCTTTCTCTGTCAAGATAACTTGAAAGAGGTTCCATTCCTCAGCTTTCGCTGGACTCAAGTGCTCAACATTCCAGCCAAATGCAACAAAATCGAAAACATCCACCAAGGCTTTGTCAACATGACTTCCCTCATCGATGTCAACTTGGGATGCAATCAAATCAGCATGGCAGCTGATACTTTCGCCAACGTTCAAGATGTCTCCAGAACTTGATTCTTGATAATAACTGCATGACTGAATTCCCAAGCAAAGCTGTGAGAAACATGAACAACTTGATTGCTCTCAAATATAACAAGATCAACGCCATTAGACAAACGACTTTGTTAACCTCTCCTCCCTCTCCATGCTCTTAATGGAAACATTTTCTTGGCTTTAAAGGAGGAGCCCTCCAGAACCATCCAAATCTTCATATCTGTATTTGAATCAGGAACAATCTGCAAACTCGACAACGGAGTCTTGGAGCAATCAAGCAACTCCTGAGGTTTCGATCTATCTTCAA

  SOLUTION: fix your fastq file

  Nov 23 14:00:36 ...... FATAL ERROR, exiting

Work dir:
  /nfs/data3/CIRCEST/runs/benchmarking/work/17/6ccdf33d1780d19e7b6bbe0973cdf6

Tip: you can replicate the issue by changing to the process work dir and entering the command `bash .command.run`

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

-[nf-core/circrna] Pipeline completed with errors-


### Relevant files


### System information

_No response_
@Fabian-Boehm Fabian-Boehm added the bug Something isn't working label Nov 23, 2023
@nictru nictru self-assigned this Nov 23, 2023
@Fabian-Boehm Fabian-Boehm changed the title Incorrect fastq reading leads to different sequence and quality string lengths. Limitted input length and inequal quality string lenths. Nov 24, 2023
@Fabian-Boehm Fabian-Boehm changed the title Limitted input length and inequal quality string lenths. Limitted input length. Nov 24, 2023
@nictru
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nictru commented Nov 24, 2023 •
edited
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Hey,
the cropping is probably only done in the error message, I can hardly imagine STAR doing something as substantial as this without giving a warning at least.

For the length inequality problem there are some related issues - maybe one of them applies to your data:

However, this problem is most likely not related to our pipeline and should thus be checked by running STAR on your data without the pipeline

@nictru
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nictru commented Jan 13, 2024

Closing this for now, feel free to re-open if the problems persist

@nictru nictru closed this as completed Jan 13, 2024
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