wf-transcriptomes-v202/docs/11_FAQ.md

Does the workflow support both cDNA and direct RNA?

Yes. Use --direct_rna for direct RNA data. cDNA is the default mode.

Do I need both --ref_genome and --ref_annotation in fixed-annotation mode?

Yes. bambu still uses the genome together with the imported annotation.

Does the workflow create one shared transcriptome or one per sample?

Both. The joint cohort model is the primary result for reporting and DE/DTU, and the workflow also emits independent per-sample transcriptomes.

Can I run DE/DTU without transcript discovery?

Yes. Use --transcriptome_mode fixed_annotation together with --de_analysis.

What changed from the previous workflow version?

The current workflow uses bambu, SQANTI3, DESeq2, and DEXSeq. The main transcriptome result is now one shared bambu model built from all samples together, with separate per-sample bambu outputs published alongside it. The most important differences are summarised in this FAQ section.

Why do the results now mention bambu and SQANTI3 instead of StringTie or GffCompare?

The workflow now uses a different set of transcript analysis tools. It builds its transcript models with bambu, classifies them with SQANTI3, and performs DE/DTU from the cohort bambu outputs.

Why is --ref_annotation now required even in fixed-annotation mode?

bambu still requires the annotation together with the genome in both discover and fixed_annotation modes. Fixed-annotation mode means annotation-driven quantification, not annotation-only execution.

Can I still use --ref_transcriptome?

No. --ref_transcriptome has been removed from the workflow interface.

Short old-to-new example:

Previous workflow version:
  --transcriptome_source precomputed --ref_transcriptome transcripts.fa

Current workflow:
  --transcriptome_mode fixed_annotation \
  --ref_genome genome.fa \
  --ref_annotation annotation.gtf

Why do I now get both cohort and per-sample transcriptomes?

The workflow now treats both as important outputs. The shared cohort model is the main transcriptome used for reporting and optional DE/DTU, while the per-sample transcriptomes are provided for looking at each sample separately.

Why are DE results under de_analysis/<contrast>/?

The workflow now writes one subdirectory per comparison instead of publishing one flat DE result set. This makes the output folder clearer when more than one comparison is present.

Short old-to-new example:

Previous workflow version:
  de_analysis/results_dge.tsv

Current workflow:
  de_analysis/<contrast>/results_dge.tsv

Can I still run fixed-annotation quantification without transcript discovery?

Yes. Use --transcriptome_mode fixed_annotation together with --ref_genome, --ref_annotation, and any optional DE/DTU settings.

What should I expect to differ in report contents and output filenames?

Expect the report and output folder to emphasise:

• the joint cohort bambu transcriptome under cohort/
• the per-sample bambu transcriptomes under samples/<alias>/
• SQANTI3 results under cohort and per-sample directories
• contrast-specific DE/DTU outputs under de_analysis/<contrast>/

If your question is not answered here, please start a discussion on the community.