Bug: Bowtie2 v2.4.1 produces malformed BAM headers causing downstream failures

[poursalavati]

Describe the bug
The pipeline fails during steps that process BAM files (e.g., the savage rule using Picard SamToFastq) with a SAMFormatException. The root cause is that the version of Bowtie2 pinned in the Conda environment (bowtie2=2.4.1) has a known bug where it creates a malformed @PG header line in the output BAM file. Specifically, the VN: (version) tag is left empty, which violates the SAM/BAM specification.

Example of the malformed header line:
@PG ID:bowtie2 PN:bowtie2 VN: CL:"..."

To Reproduce
Steps to reproduce the behavior:

1. V-pipe configuration file used: Any configuration where aligner: bowtie2 is selected.
2. Samples TSV file used: Any standard paired-end FASTQ samples.
3. Commands executed: Run the pipeline past the alignment stage, e.g., snakemake --use-conda --cores 32
4. See error: The error will manifest as a SAMFormatException in a later rule that uses a strict BAM parser like Picard. The cause can be seen immediately by inspecting the header of the alignment BAM file: samtools view -H path/to/results/sample/alignments/REF_aln.bam.

Expected behavior
The BAM file generated by the Bowtie2 alignment step should have a valid header. The @PG line for bowtie2 should contain a correctly formatted version number, for example: VN:2.5.1. The pipeline should not fail in downstream steps due to header validation errors.

Screenshots
N/A

Desktop (please complete the following information):

• OS: Linux (HPC environment)
• Version: master branch

Additional context
This issue is completely resolved by updating the Bowtie2 dependency to a more recent version where this bug is fixed. I can confirm that changing the environment file workflow/envs/bowtie2.yaml to the following resolves the issue:

channels:
  - conda-forge
  - bioconda
dependencies:
  - bowtie2>=2.5.0
  - samtools=1.10

This ensures that all generated alignment files are valid and prevents errors in downstream processing.

[gordonkoehn]

Thanks for the clear report and the suggested fix — very helpful. I'll add @DrYak to investigate.