Pipe line Error with HiSAT2 Aligner

[merang2050]

Description of the bug

I have some errors when I run the pipe line with HiSAT2 aligner. If I use same configuration with STAR RSEM, I don't have any errors.

Command used and terminal output

nextflow run nf-core/rnaseq -r 3.14.0 \
--outdir iBOSK_HiSAT2_results \
-c /resources/nf_hisat2.config \
-profile singularity \
--input /iBOSK_sheetList.csv \
--aligner hisat2 \
--igenomes_ignore \
--genome null \
--multiqc_title "iBOSK HiSAT2 RNA-seq" \
--gencode \
--fasta GRCm38/GRCm38.primary_assembly.genome.fa.gz \
--gtf GRCm38/gencode.vM25.annotation.gtf.gz \




Errors : 

-        ] process > NFCORE_RNASEQ:RNASEQ:BAM_RSEQC:RSEQC_READDUPLICATION                                                        -
[-        ] process > NFCORE_RNASEQ:RNASEQ:CUSTOM_DUMPSOFTWAREVERSIONS                                                            -
[-        ] process > NFCORE_RNASEQ:RNASEQ:MULTIQC                                                                                -
[HISAT2 index build] Available memory: 128 GB
[HISAT2 index build] Less than 200 GB available, so NOT using splice sites and exons to build HISAT2 index.
[HISAT2 index build] Use --hisat2_build_memory [small number] to skip this check.
Execution cancelled -- Finishing pending tasks before exit
ERROR ~ Error executing process > 'NFCORE_RNASEQ:RNASEQ:FASTQ_ALIGN_HISAT2:HISAT2_ALIGN (iF_Novo_56)'

Caused by:
  Process `NFCORE_RNASEQ:RNASEQ:FASTQ_ALIGN_HISAT2:HISAT2_ALIGN (iF_Novo_56)` terminated with an error exit status (1)

Command executed:

  INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\.1.ht2$//'`
  hisat2 \
      -x $INDEX \
      -1 iF_Novo_56_1_val_1.fq.gz \
      -2 iF_Novo_56_2_val_2.fq.gz \
       \
      --known-splicesite-infile GRCm38.primary_assembly.genome.filtered.splice_sites.txt \
      --summary-file iF_Novo_56.hisat2.summary.log \
      --threads 12 \
      --rg-id iF_Novo_56 --rg SM:iF_Novo_56 \
       \
      --no-mixed \
      --no-discordant \
      --met-stderr --new-summary --dta \
      | samtools view -bS -F 4 -F 8 -F 256 - > iF_Novo_56.bam
  
  if [ -f iF_Novo_56.unmapped.fastq.1.gz ]; then
      mv iF_Novo_56.unmapped.fastq.1.gz iF_Novo_56.unmapped_1.fastq.gz
  fi
  if [ -f iF_Novo_56.unmapped.fastq.2.gz ]; then
      mv iF_Novo_56.unmapped.fastq.2.gz iF_Novo_56.unmapped_2.fastq.gz
  fi
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_RNASEQ:RNASEQ:FASTQ_ALIGN_HISAT2:HISAT2_ALIGN":
      hisat2: 2.2.1
      samtools: $(echo $(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*$//')
  END_VERSIONS

Command exit status:
  1

Command output:
  (empty)

Command error:
  INFO:    Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred
  INFO:    Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred
  INFO:    Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred
  WARNING: Skipping mount /var/lib/apptainer/mnt/session/etc/resolv.conf [files]: /etc/resolv.conf doesn't exist in container
  (ERR): mkfifo(/tmp/101.inpipe1) failed.
  Exiting now ...
  [main_samview] fail to read the header from "-".

Work dir:
  /mnt/beegfs/shares/tian_lab/shared/database/iBOSK_rnaseq_results/work/00/9daac2088f5fe8b8fd92cfff03c833

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

[merang2050]

memory issue is solved but still have this error :

[- ] process > NFCORE_RNASEQ:RNASEQ:MULTIQC - Execution cancelled -- Finishing pending tasks before exit ERROR ~ Error executing process > 'NFCORE_RNASEQ:RNASEQ:FASTQ_ALIGN_HISAT2:HISAT2_ALIGN (iF_Novo_3)'

Caused by: Process NFCORE_RNASEQ:RNASEQ:FASTQ_ALIGN_HISAT2:HISAT2_ALIGN (iF_Novo_3) terminated with an error exit status (1)

Command executed:

INDEX=find -L ./ -name "*.1.ht2" | sed 's/\.1.ht2$//' hisat2
-x $INDEX
-1 iF_Novo_3_1_val_1.fq.gz
-2 iF_Novo_3_2_val_2.fq.gz

--known-splicesite-infile GRCm38.primary_assembly.genome.filtered.splice_sites.txt
--summary-file iF_Novo_3.hisat2.summary.log
--threads 12
--rg-id iF_Novo_3 --rg SM:iF_Novo_3

--no-mixed
--no-discordant
--met-stderr --new-summary --dta
| samtools view -bS -F 4 -F 8 -F 256 - > iF_Novo_3.bam

if [ -f iF_Novo_3.unmapped.fastq.1.gz ]; then mv iF_Novo_3.unmapped.fastq.1.gz iF_Novo_3.unmapped_1.fastq.gz fi if [ -f iF_Novo_3.unmapped.fastq.2.gz ]; then mv iF_Novo_3.unmapped.fastq.2.gz iF_Novo_3.unmapped_2.fastq.gz fi

cat <<-END_VERSIONS > versions.yml "NFCORE_RNASEQ:RNASEQ:FASTQ_ALIGN_HISAT2:HISAT2_ALIGN": hisat2: 2.2.1 samtools: $(echo $(samtools --version 2>&1) | sed 's/^.samtools //; s/Using.$//') END_VERSIONS

Command exit status: 1

Command output: (empty)

Command error: INFO: Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred WARNING: Skipping mount /var/lib/apptainer/mnt/session/etc/resolv.conf [files]: /etc/resolv.conf doesn't exist in container (ERR): mkfifo(/tmp/101.inpipe1) failed. Exiting now ... [main_samview] fail to read the header from "-".

Work dir: database/iBOSK_rnaseq_results/work/56/0cd01a76631b2a192eaf55b592a649

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

[pinin4fjords]

I think it's quite likely HISAT2 is failing for some reason with those input data, which is causing the pipe error, see this Slack thread: https://nfcore.slack.com/archives/CE8SSJV3N/p1713885189645209. I would start with the actions proposed there.

[drpatelh]

Closing for now but feel free to re-open if required. See https://github.com/nf-core/rnaseq/issues/1270#issuecomment-2178088070