Chapter 6 Run the Salmon and STAR workflows

6.1 Run Salmon mode runall

iobrpy runall \
  --mode salmon \
  --outdir "$IOBRPY_DEMO/results_salmon" \
  --fastq "$IOBRPY_DEMO/input/fastq" \
  --threads 8 \
  --batch_size 1 \
  --index "$IOBRPY_REF/salmon/gencode44" \
  --project PRJNA1161405

6.2 Run STAR mode runall

iobrpy runall \
  --mode star \
  --outdir "$IOBRPY_DEMO/results_star" \
  --fastq "$IOBRPY_DEMO/input/fastq" \
  --threads 8 \
  --batch_size 1 \
  --index "$IOBRPY_REF/star" \
  --project PRJNA1161405

6.3 What is already included

Do not rerun these components in separate chapters. Both runall modes already perform:

  1. fastp quality control;
  2. Salmon quantification or STAR alignment and gene counting;
  3. merged expression output, TPM matrix preparation, and log2 transformation of the TPM matrix;
  4. signature scoring;
  5. six-method TME panel: run CIBERSORT, IPS, ESTIMATE, MCPcounter, quanTIseq, and EPIC, then merge their outputs;
  6. ligand-receptor scoring;
  7. TRUST4 TCR/BCR analysis.

6.4 Salmon mode output contract

$IOBRPY_DEMO/results_salmon/
├── 01-qc/
├── 02-salmon/
│   ├── <sample>/quant.sf
│   ├── PRJNA1161405_salmon_count.tsv.gz
│   └── PRJNA1161405_salmon_tpm.tsv.gz
├── 03-tpm/
│   ├── prepare_salmon.csv
│   └── tpm_matrix.csv
├── 04-signatures/calculate_sig_score.csv
├── 05-tme/
│   ├── cibersort_results.csv
│   ├── IPS_results.csv
│   ├── estimate_results.csv
│   ├── mcpcounter_results.csv
│   ├── quantiseq_results.csv
│   ├── epic_results.csv
│   └── deconvo_merged.csv
├── 06-LR_cal/lr_cal.csv
└── 07-TCRBCR/
    ├── <sample>/<sample>_report.tsv
    ├── trust4_immdata.csv
    └── trust4_immune_indices.csv

6.5 STAR mode output contract

$IOBRPY_DEMO/results_star/
├── 01-qc/
├── 02-star/
│   ├── <sample>_Aligned.sortedByCoord.out.bam
│   ├── <sample>_ReadsPerGene.out.tab
│   └── PRJNA1161405.STAR.count.tsv.gz
├── 03-tpm/
│   ├── count2tpm.csv
│   └── tpm_matrix.csv
├── 04-signatures/calculate_sig_score.csv
├── 05-tme/
│   ├── cibersort_results.csv
│   ├── IPS_results.csv
│   ├── estimate_results.csv
│   ├── mcpcounter_results.csv
│   ├── quantiseq_results.csv
│   ├── epic_results.csv
│   └── deconvo_merged.csv
├── 06-LR_cal/lr_cal.csv
└── 07-TCRBCR/
    ├── <sample>/<sample>_report.tsv
    ├── trust4_immdata.csv
    └── trust4_immune_indices.csv

The standalone analyses chapter reuses three outputs:

  • 03-tpm/count2tpm.csv for BayesPrism;
  • 05-tme/cibersort_results.csv for TME and NMF clustering;
  • 02-star/*_Aligned.sortedByCoord.out.bam for HLA typing.