rnaseq completeness audit (2026-08-21)
Upstream: nf-core/rnaseq @ 3.26.0 · Port: oxo-flow-rnaseq (live-verified).
Mode matrix (upstream — one workflow, parameter cross-product)
| axis | options | default |
|---|---|---|
| aligner | star_salmon / star_rsem / hisat2 / bowtie2_salmon | star_salmon |
| pseudo-aligner (add-on) | salmon / kallisto (null=off) | off |
| trimmer | trimgalore / fastp | trimgalore |
| BAM-input mode | --skip_alignment + BAM samplesheet columns |
— |
| UMI | --with_umi + --umi_dedup_tool umitools/umicollapse |
off |
| rRNA removal | --remove_ribo_rna + sortmerna/ribodetector/bowtie2 |
off |
| contaminant screening | kraken2 / kraken2_bracken / sylph | off |
| QC engine | default multi-tool / --use_rustqc |
default |
| STAR engine | OSS / --use_sentieon_star / --use_parabricks_star |
OSS |
| prokaryotic | --prokaryotic (+bowtie2_salmon, CDS featureCounts) |
off |
No differential-analysis workflow at this tag (DESeq2 QC = PCA/clustering only). UHR/UMI legacy params removed.
Gap tiers vs the port
P0 (portable): hisat2 branch (extract_splicesites + build + align — no BAM quantification downstream); star_rsem branch (RSEM prepare-reference + calculateexpression + merge + tximport); bowtie2_salmon (+prokaryotic config); pseudo-alignment add-on (salmon/kallisto quant + tximport); BAM-input mode; UMI mode (extract + dedup_umitools/umicollapse + prepareforrsem); rRNA removal (3 tools); contaminant screening (kraken2/bracken/sylph); fastp trimmer variant; stringtie ignore-gtf merge mode.
P1 (license): sentieon (paid license secret), parabricks (commercial NGC container + GPU), SILVA-based SortMeRNA default DB (non-academic license — bowtie2/ribodetector are the license-free routes; document).
P2 (config): all --skip_* QC gates, --rseqc_modules list,
--bam_csi_index, strand thresholds, quant libtype args,
--save_* publish gates, --stringtie_ignore_gtf, --gencode,
--skip_quantification_merge, --min_mapped_reads, extra-tool args,
iGenomes.
External deps: iGenomes S3, nf-core/configs fetch, quay.io + Seqera Wave containers, test data HTTPS.
Verdict
Large P0 surface: 2 aligner backends + 2 add-on quantifiers + 4 orthogonal feature modes (UMI/rRNA/contaminant/BAM-input) beyond the ported star_salmon default.
Re-verification (2026-08-23, heavy group 3/3 — heavy 收官)
Engine: latest main (post-v0.14.1) · Box: tx-ubuntu · Mode: real CLI run, not dry-run (checkpoint cleared, forced real execution).
Cumulative 68 rules really executed, 0 real failures — first run 65/15/2 (both failures were env-build: genomeinfodbdata post-link proxy truncation + the deseq2 name collision), then a resume chain: TLS-local conda CA fix → tximport → deseq2 env rebuild (r-optparse added) → deseq2_qc → final 2/80/0 exit 0. Coverage chain (real): fastp → STAR+quantMode → biotype → qualimap → dupradar → bigwig fw/rev/combined → tximport → summarizedexperiment → deseq2_qc → multiqc. All box-side lessons cataloged (stale env-cache / cross-repo name collision → issue #159 / post-link proxy truncation).