TUSCO (Transcriptome Universal Single‑isoform Control) provides a compact, curated set of single‑isoform genes to benchmark transcriptome reconstruction. When enabled in SQANTI3 QC, it produces a self‑contained HTML report summarizing detection performance and per‑gene visualizations (Gviz‑based, IGV‑like tracks). This guide specifies inputs, execution, outputs, and interpretation to support rigorous and reproducible use.

• Isoform annotations: provide a transcript GTF via --isoforms for optimal plotting.
  • If starting from FASTA/FASTQ, SQANTI3 generates a corrected GTF internally, which is then used by TUSCO.
• Reference resources: --refGTF (gene annotation) and --refFasta (genome assembly) are required.
• Reporting: ensure --report is not skipped; TUSCO runs alongside the standard SQANTI3 QC report.

python sqanti3_qc.py \
  --isoforms your_transcripts.gtf \
  --refGTF reference.gtf \
  --refFasta genome.fasta \
  --tusco human|mouse \
  --report html \
  -o <prefix> -d <outdir>

Key flag:

• --tusco: selects the bundled species panel (human or mouse).

• (<outdir>)/(<prefix>)_TUSCO_report.html — self‑contained HTML benchmarking report.
• (<outdir>)/logs/tusco_report.log — Rscript log for the TUSCO report generation.
• (<outdir>)/igv_plots/ — one PNG per TUSCO gene with Gviz tracks and SQANTI3 category coloring.

The TUSCO report is optional. If it fails, the main QC pipeline continues. Consult tusco_report.log for diagnostics.

The repository ships with human and mouse TUSCO panels as TSV files:

• Human: src/utilities/report_qc/tusco_human.tsv
• Mouse: src/utilities/report_qc/tusco_mouse.tsv

Additional details:

• Current gene counts: human = 50, mouse = 32.
• Assembly mapping for plots: human → hg38; mouse → mm10.

Note:

• Only the TSV file is required for TUSCO operation. Files are named tusco_<species>.tsv.

Curated tissue‑specific TUSCO references for human and mouse are available at https://tusco.uv.es.

To use a tissue‑specific panel:

1. Obtain the TSV for your tissue and species.
2. Replace the bundled TSV in src/utilities/report_qc/ for that species, keeping filenames unchanged:
   • Human: tusco_human.tsv
   • Mouse: tusco_mouse.tsv
3. Run SQANTI3 QC with --tusco human or --tusco mouse.

TUSCO summarizes detection performance across its single‑isoform gene set and provides per‑gene visualizations. The report presents six primary metrics to maintain a “higher‑is‑better” orientation. Let TP/FP/FN be counts within the TUSCO set; nr denotes non‑redundant (gene‑level) counting; r denotes redundant (transcript‑level) counting.

Metrics (canonical definitions):

Notes:

• The HTML dashboard labels follow the symbols above (Sn, nrPre, rPre, 1−FDR, PDR, 1/red). Redundancy itself can be inspected in detailed tables/plots; 1/red is shown to align directionality.
• Exact implementation follows the SQANTI3 TUSCO report; the symbols/formulas above are provided for interpretation.

Classification categories used in the report:

• TP (True Positive): reference match or qualifying mono‑exon Full Splice Match (FSM).
• PTP (Partial True Positive): FSM/Incomplete Splice Match (ISM) not counted as reference match (RM).
• FP (False Positive): non‑canonical categories within the TUSCO set (e.g., NIC, NNC, genic, antisense, fusion, intergenic).
• FN (False Negative): TUSCO genes with no detected transcript.

Per‑gene plots:

• Sample transcripts are colored by SQANTI3 structural category (e.g., FSM, ISM, NIC, NNC).

For definitions of SQANTI3 categories, see: https://github.com/ConesaLab/SQANTI3/wiki/SQANTI3-isoform-classification:-categories-and-subcategories

Support for additional species requires code changes to recognize a new --tusco value and to define the corresponding genome assembly for Gviz plotting.

• Practical workaround: rename your custom TSV to tusco_human.tsv, then run with --tusco human (plots will use hg38 conventions).

• TUSCO evaluates detection and correctness within a curated set of single‑isoform genes; it does not substitute for comprehensive transcriptome‑wide evaluation.
• Metrics are panel‑relative and reflect the behavior on well‑behaved genes; interpret alongside broader QC and task‑specific assessments.

• Missing or empty report: check (<outdir>)/logs/tusco_report.log for Rscript errors.
• No or sparse plots: provide --isoforms as a GTF if possible; confirm --refGTF/--refFasta match the expected assemblies (human → hg38, mouse → mm10).
• Panel mismatch: ensure the .tsv was correctly replaced when switching to a tissue‑specific set.
• Environment: the report uses R with Gviz‑based plotting; ensure required R packages are available if you are running outside the standard setup.
• Reproducibility checklist: record SQANTI3 commit/version, panel file checksums (tusco_<species>.tsv), command lines, and the tusco_report.log. Use fixed RNG seeds where applicable.

• TUSCO‑novel (Novel Isoform Stress Test) — methodology to stress‑test novel discovery; uses the same TSV panel and TUSCO metrics.

If TUSCO is used in analyses or figures, please cite SQANTI3 and the TUSCO resource as appropriate. Include software versions/commits, panel provenance (species, date, and filename), and commands sufficient to reproduce the report.