--clip3pAdapterSeq has to contain 2 values to match the number of mates

[SuhasSrinivasan]

Description of the bug

Dear Developers,

Thank you for developing and maintaining the pipeline.

When following the instructions for 3′ digital gene expression assays there is a consistent issue when specifying the custom Star arguments for paired end sequencing.

Pipeline completed with errors-
ERROR ~ Error executing process > 'NFCORE_RNASEQ:RNASEQ:ALIGN_STAR:STAR_ALIGN (D0_2)'

Caused by:
  Process `NFCORE_RNASEQ:RNASEQ:ALIGN_STAR:STAR_ALIGN (D0_2)` terminated with an error exit status (102)


Command executed:

  STAR \
      --genomeDir star \
      --readFilesIn input1/D0_2_trimmed_1_val_1.fq.gz input2/D0_2_trimmed_2_val_2.fq.gz \
      --runThreadN 12 \
      --outFileNamePrefix D0_2. \
       \
      --sjdbGTFfile GRCh38.primary_assembly.genome.filtered.gtf \
      --outSAMattrRGline 'ID:D0_2'  'SM:D0_2'  \
      --quantMode TranscriptomeSAM --twopassMode Basic --outSAMtype BAM Unsorted --readFilesCommand zcat --runRNGseed 0 --outFilterMultimapNmax 20 --alignSJDBoverhangMin 1 --outSAMattributes NH HI AS NM MD --outSAMstrandField intronMotif --alignIntronMax 1000000 --alignIntronMin 20 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --outFilterMismatchNmax 999 --outFilterType BySJout --outFilterMismatchNoverLmax 0.1 --clip3pAdapterSeq AAAAAAAA --quantTranscriptomeSAMoutput BanSingleEnd



  if [ -f D0_2.Unmapped.out.mate1 ]; then
      mv D0_2.Unmapped.out.mate1 D0_2.unmapped_1.fastq
      gzip D0_2.unmapped_1.fastq
  fi
  if [ -f D0_2.Unmapped.out.mate2 ]; then
      mv D0_2.Unmapped.out.mate2 D0_2.unmapped_2.fastq
      gzip D0_2.unmapped_2.fastq
  fi

  cat <<-END_VERSIONS > versions.yml
  "NFCORE_RNASEQ:RNASEQ:ALIGN_STAR:STAR_ALIGN":
      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:
  102

Command output:
  (empty)

Command error:

  EXITING because of fatal PARAMETER error: --clip3pAdapterSeq has to contain 2 values to match the number of mates.
  SOLUTION: specify 2values in --clip3pAdapterSeq , for no clipping use -
  Apr 20 18:26:24 ...... FATAL ERROR, exiting

Workaround

--extra_star_args "--clip3pAdapterSeq AAAAAAAA AAAAAAAA"

Command used and terminal output

nextflow run nf-core/rnaseq 
-profile singularity 
--save_reference \
--input /mnt/storage/projects/r2/rna_initial/fastqs/samples.csv \
--outdir  /mnt/storage/projects/r2/rna_initial/ \
--gtf /mnt/storage/projects/reference/rna/gencode.v47.primary_assembly.annotation.gtf \
--fasta /mnt/storage/projects/reference/rna/GRCh38.primary_assembly.genome.fa \
--extra_star_align_args "--alignIntronMax 1000000 --alignIntronMin 20 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --outFilterMismatchNmax 999 --outFilterMultimapNmax 20 --outFilterType BySJout --outFilterMismatchNoverLmax 0.1 --clip3pAdapterSeq AAAAAAAA" \
--extra_salmon_quant_args="--noLengthCorrection"

Relevant files

No response

System information

Version of nf-core/rnaseq: 3.18.0
Nextflow version: v24.10.5
Container engine: singularity-ce version 4.1.2-jammy
OS: Ubuntu 22.04.5 LTS x86_64
Executor: Local
Hardware: Desktop

[pinin4fjords]

Could you clarify please- you clearly have a working version, what is the issue? Could you highlight any documentation you feel is incorrect and suggest a fix?

[SuhasSrinivasan]

Hi @pinin4fjords
Thank you for reviewing this and the previous issues.

When using paired-end sequencing with Lexogen QuantSeq 3‘ mRNA-Seq FWD, the Custom STAR parameters in the docs need an update.

--extra_star_args "--clip3pAdapterSeq AAAAAAAA AAAAAAAA"

[pinin4fjords]

Actually, I suspect the issue here (and in the other issue) is that the documentation was written for the typical 3' DGE use case, where data are expected to be single-end. So I'm disinclined to change the documentation for a paired-end use case.

Could you confirm which assay you're using please, and why your data are paired end?

[SuhasSrinivasan]

Hi,

We used Lexogen's QuantSeq 3' mRNA-Seq FWD.

Link to manual

Although pairedend sequencing is possible, we do not recommend it for QuantSeq FWD. Read 2 would start with the poly(T) stretch, and sequence through the homopolymer stretch, reducing the quality of Read 2.

[pinin4fjords]

OK, so sounds like sticking with the assumption of single-endedness, per the current docs, is sensible.

[SuhasSrinivasan]

Thank you!