100 lines
4.4 KiB
Markdown
100 lines
4.4 KiB
Markdown
+ Check that the reference genome and reference annotation use overlapping
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sequence names. The workflow checks this early and will fail if there is no
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overlap at all.
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+ If `--de_analysis` is enabled, ensure the sample sheet contains `alias`, the
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primary condition column, and any columns named in `--covariates`.
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+ DE/DTU requires at least two condition levels and at least two samples per
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level.
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+ `--cdna_preprocess` must not be combined with `--direct_rna`.
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+ See how to interpret common Nextflow exit codes
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[here](https://labs.epi2me.io/trouble-shooting/).
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### Common confusion when coming from the previous workflow version
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#### I supplied `--ref_transcriptome`, but the workflow still built or used `bambu` outputs
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The previous workflow version used `--ref_transcriptome` as a main driver for
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transcript-level downstream analysis. In the current version the
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`--ref_transcriptome` option has been removed and is not part of the current
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`bambu` input setup.
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Use `--transcriptome_mode fixed_annotation` together with
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`--ref_genome` and `--ref_annotation` if you want annotation-driven quantification
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without transcript discovery.
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#### I omitted `--ref_annotation` because I expected the old input setup
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The previous workflow version could be driven from a different combination of
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transcriptome inputs. In the current version both `--ref_genome` and
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`--ref_annotation` are required in `discover` and `fixed_annotation` modes.
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Always provide a compatible genome FASTA and transcript annotation when
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launching the workflow.
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#### I used `--transcriptome_source` and got behaviour I did not expect
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In the previous workflow version the `--transcriptome_source` was the main
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setting that chose how the workflow behaved. In the current version,
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`--transcriptome_mode` is the main setting that controls this. The
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`--transcriptome_source` option has been removed from the workflow interface.
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The options `--transcriptome_mode discover` or `--transcriptome_mode
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fixed_annotation` should be used to choose between the modes of operation.
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#### I expected pychopper-style preprocessing for cDNA libraries
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cDNA preprocessing is available through
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`--cdna_preprocess`, which runs `pychopper` before alignment. The
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parameters for that stage are `--cdna_kit`, `--pychopper_backend`, and
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`--pychopper_opts`.
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Enable `--cdna_preprocess` for cDNA libraries when you want
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`pychopper` preprocessing, or use `--direct_rna` without
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`--cdna_preprocess` for direct RNA data.
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#### I cannot find the old flat DE output files
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In the previous workflow version, DE files appeared directly under `de_analysis/`.
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In the current version DE and DTU results are grouped by contrast under
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`de_analysis/<contrast>/`. Look for outputs such as
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`de_analysis/<contrast>/results_dge.tsv` and
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`de_analysis/<contrast>/results_dtu_transcript.tsv`.
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#### I expected the old output layout or transcriptome files
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The previous workflow version emitted one flat set of transcriptome.
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In the current versions, the output folder is organised around `ingress_results/`,
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`cohort/`, `samples/<alias>/`, `de_analysis/<contrast>/`, and
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`igv_reference/`.
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Primary shared transcriptome results are under `cohort/`, and
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`samples/<alias>/` for sample-specific models. If `--cdna_preprocess` is enabled,
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look under `ingress_results/<alias>/` for the `pychopper` outputs associated
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with each sample.
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#### My DE/DTU run fails because of `--sample_sheet`
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The sample sheet rules have been amended compared to the previous version
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to allow for multi-way comparisons.
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The sample sheet must contain `alias`, the primary condition
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column, and any columns named in `--covariates`. At least two condition levels
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are required, and each level must contain at least two samples.
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What to change: verify the sample sheet columns first, then check
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`--condition_column`, `--covariates`, and `--reference_level`.
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#### I combined `--direct_rna` with `--cdna_preprocess`
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This is not a valid combination of parameters. Use `--direct_rna` for direct RNA
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libraries and leave `--cdna_preprocess` unset.
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#### I hit genome/annotation validation or strand-related annotation warnings
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The workflow validates that the annotation and genome share
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sequence names, and it excludes unstranded annotation entries from the
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differential analysis path.
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Confirm the genome and annotation come from a compatible source,
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and ensure the annotation uses only `+` or `-` strand values where required for
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DE/DTU and `SQANTI3`.
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