RNA-seq Quantification — salmon & kallisto Transcript Abundance

The command-line quantification entry point for bulk RNA-seq: take raw FASTQ reads plus a reference transcriptome and produce transcript-level abundance estimates (counts + TPM) with salmon (selective alignment) or kallisto (pseudoalignment via kb-python), then aggregate to the gene level with tximport/tximeta and hand off to alterlab-pydeseq2 for differential expression. It is the raw-data-to-count-matrix pipeline that the repo's Python analysis skills assume already ran.

Quick Start

Quantify these RNA-seq FASTQs with salmon and a decoy-aware index
Build a salmon gentrome index from this transcriptome + genome
Run kallisto / kb count on my paired-end reads
Turn my salmon quant.sf files into a gene-level count matrix for DESeq2

→ Build a decoy-aware index once, run salmon quant (or kb count) per sample, then run scripts/build_tx2gene.py + scripts/import_quant.py to make the tximport gene matrix and route it to alterlab-pydeseq2.

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When to Use This Skill

Use this skill when the request is about getting from FASTQ to transcript or gene abundance with a lightweight quantifier:

• "Quantify my RNA-seq with salmon / kallisto."
• "Build a decoy-aware salmon index (gentrome + decoys.txt)."
• "Run selective alignment with --validateMappings --gcBias."
• "I have quant.sf files — make me a gene-level count matrix for DESeq2."
• "Set up tximport / tximeta with a tx2gene map."
• "Use kb-python / kb count to pseudoalign these reads."

Does NOT Trigger — route these to the right sibling

The request is really about…	Route to
Differential expression on a count matrix (DESeq2 Wald tests, FDR, volcano)	alterlab-pydeseq2
Single-cell RNA-seq quantification (was salmon alevin)	piscem + alevin-fry — see references/single_cell_alevin.md; downstream → alterlab-scanpy / alterlab-scvi-tools
FASTQ-to-VCF germline/somatic variant calling	alterlab-nf-core-sarek
16S/ITS amplicon (microbiome) FASTQ-to-feature-table	alterlab-qiime2-amplicon
Spatial transcriptomics (Visium/Xenium) neighborhood analysis	alterlab-squidpy-spatial
Loading/manipulating the resulting matrix as an AnnData object	alterlab-anndata
BLAST/DIAMOND sequence similarity search	alterlab-blast
Quick gene/transcript ID lookups & reference fetch (Ensembl/RefSeq)	alterlab-gget
Aligned BAM manipulation, coverage, read counting from alignments	alterlab-pysam

This skill stops at the count/abundance matrix. It does not call DEGs, does not handle single-cell barcodes, and does not align to a genome for variant calling.

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Two Critical Correctness Traps (read before quantifying)

These are the two failures most outdated RNA-seq instructions get wrong as of salmon v1.11.4 (released 2026-03-11). Both are confirmed in the upstream release notes (see references/tool_versions.md).

1. The salmon index format changed to SSHash. salmon switched from the colored compacted de Bruijn graph index to a new SSHash-based k-mer index. The release notes state all previously built indices must be rebuilt before using v1.11.2+. If you reuse a pre-v1.11.2 index you will get an error or silently wrong results — always rebuild the index with the same salmon version you quantify with.

2. salmon alevin was REMOVED. Single-cell quantification is no longer part of salmon. The release notes direct former alevin users to the piscem + alevin-fry pipeline. Do not write salmon alevin commands. If the user has single-cell / droplet data, route per the table above and see references/single_cell_alevin.md.

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Pipeline (salmon, the default path)

1. Build a decoy-aware gentrome index (once per reference)

A decoy-aware index lets salmon distinguish reads that align better to the genome than the transcriptome, reducing spurious assignments. You build a "gentrome" = transcripts FASTA concatenated with the genome FASTA, plus a decoys.txt listing the genome sequence names as decoys.

# 1. decoys.txt = the genome's sequence (chromosome) names, one per line
grep "^>" genome.fa | sed 's/^>//; s/ .*//' > decoys.txt

# 2. gentrome = transcripts FIRST, then genome (order matters)
cat transcripts.fa genome.fa > gentrome.fa

# 3. build the index (rebuild for v1.11.4 — see trap #1)
salmon index \
  -t gentrome.fa \
  -d decoys.txt \
  -i salmon_index \
  -k 31 \
  -p 8

• -k 31 is the default k-mer; lower it only for very short reads.
• The helper scripts/make_decoys.py writes decoys.txt and gentrome.fa for you and refuses to proceed if the genome names are absent from the transcript FASTA (a common silent mistake). See references/decoy_index.md.

2. Quantify each sample

salmon quant \
  -i salmon_index \
  -l A \
  -1 sampleA_R1.fastq.gz -2 sampleA_R2.fastq.gz \
  --validateMappings \
  --gcBias \
  -p 8 \
  -o quants/sampleA

• -l A — auto-detect library type (strandedness). Let salmon infer it unless you have a documented protocol; verify the inferred type in lib_format_counts.json.
• --validateMappings — enables selective alignment (the accurate default mode; scores mappings rather than trusting raw pseudo-mappings).
• --gcBias — corrects fragment-level GC bias; recommended for DE and cheap to enable. Add --seqBias for 5'/3' sequence-specific bias if needed.
• For single-end reads, pass -r reads.fastq.gz instead of -1/-2.

Each sample produces quants/<sample>/quant.sf (transcript-level estimates) and quants/<sample>/lib_format_counts.json (the inferred library type). See references/salmon_quant.md for the full flag map and per-sample QC checks.

3. Aggregate to gene level with tximport

Build a transcript→gene map (tx2gene) from your annotation, then summarize the per-sample quant.sf files into a gene-level matrix that pydeseq2 consumes.

# tx2gene from a GTF/GFF3 (transcript_id -> gene_id)
uv run python scripts/build_tx2gene.py annotation.gtf --out tx2gene.tsv

# import + summarize to gene level (tximport "lengthScaledTPM" counts)
uv run python scripts/import_quant.py \
  --quants quants \
  --tx2gene tx2gene.tsv \
  --out-counts gene_counts.tsv \
  --out-tpm gene_tpm.tsv

import_quant.py produces an integer-rounded gene × sample count matrix plus a gene × sample TPM matrix, the inputs alterlab-pydeseq2 expects. It implements tximport's makeCountsFromAbundance(..., "lengthScaledTPM") at the transcript level (scale each transcript's TPM by its sample-averaged effective length, then rescale each sample column back to its mapped-read library size) and sums to genes — so the counts are length-corrected and library-size-scaled, not raw summed NumReads. The canonical R route is the tximport / tximeta Bioconductor packages with countsFromAbundance = "lengthScaledTPM"; the Python helper here reproduces that computation so you can stay in uv (differing only in integer rounding and the absence of tximeta provenance). See references/tximport_handoff.md for the exact semantics, the tximeta linkedTxome metadata option, and when to prefer the R path.

4. Hand off to differential expression

Pass gene_counts.tsv (+ a sample/condition sheet) to alterlab-pydeseq2. This skill does not call DEGs — that is pydeseq2's job (size-factor normalization, dispersion, Wald tests, BH-FDR, volcano/MA plots).

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Pipeline (kallisto, the pseudoalignment path)

kallisto (standalone v0.52.0) and the kb-python wrapper (kb) give a faster pseudoalignment route. kb-python drives kallisto | bustools and writes tidy outputs.

# build a kallisto index from the transcriptome
kallisto index -i kallisto_index.idx transcripts.fa

# quantify a paired-end sample
kallisto quant -i kallisto_index.idx -o quants_kallisto/sampleA \
  sampleA_R1.fastq.gz sampleA_R2.fastq.gz

# OR the kb-python workflow (bulk)
# -f1 is the cDNA FASTA kb WRITES; trailing positionals are genome FASTA THEN GTF
kb ref -i index.idx -g t2g.txt -f1 cdna.fa genome.fa annotation.gtf
kb count -i index.idx -g t2g.txt -x bulk -o quants_kb/sampleA \
  sampleA_R1.fastq.gz sampleA_R2.fastq.gz

• kallisto outputs abundance.tsv / abundance.h5; feed these to tximport (type="kallisto") the same way as salmon's quant.sf.
• Long reads: kb-python exposes lr-kallisto via the --long flag (and k>31 k-mers) — use it for ONT/PacBio cDNA. See references/kallisto_kb.md.
• kallisto does not use the decoy/gentrome construction; that is salmon-specific.

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Turnkey alternative — nf-core/rnaseq

For an end-to-end, provenance-tracked pipeline (trimming → alignment → quantification → QC), nf-core/rnaseq v3.26.0 runs --aligner star_salmon by default: STAR maps to the genome, projects onto the transcriptome, and Salmon does the quantification. Reach for it when the user wants a reproducible Nextflow pipeline rather than hand-run commands; this skill covers the direct-salmon/kallisto path and the tximport handoff. See references/tool_versions.md.

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Offload note

Indexing and per-sample quantification are CPU/IO-heavy but fully offline. On a local workstation these are good candidates to run directly (e.g. overnight) rather than streaming large FASTQs through an API session. Build the index once; quantify samples in a loop.

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Self-Check Before Reporting

• Did you rebuild the salmon index with the v1.11.4 you quantified with (SSHash format — trap #1)? Never reuse a pre-v1.11.2 index.
• Is the index decoy-aware (gentrome + decoys.txt) for salmon? Confirm the genome names made it into decoys.txt.
• Did you let -l A infer strandedness, and did you sanity-check the inferred type in lib_format_counts.json?
• Is the data actually single-cell? If so you must NOT use this path — salmon alevin is gone; route to piscem + alevin-fry (trap #2).
• Did you stop at the count matrix and hand DE off to alterlab-pydeseq2 rather than calling DEGs here?

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References

• references/tool_versions.md — pinned versions (salmon v1.11.4, kallisto v0.52.0, kb-python, nf-core/rnaseq v3.26.0) and the upstream release-note facts (SSHash index change, alevin removal).
• references/decoy_index.md — decoy-aware gentrome index construction, gotchas, and the make_decoys.py helper.
• references/salmon_quant.md — salmon quant flag map, library-type inference, and per-sample QC.
• references/kallisto_kb.md — kallisto / kb-python workflow, --long (lr-kallisto), and output handling.
• references/tximport_handoff.md — tximport / tximeta aggregation, tx2gene, countsFromAbundance, and the pydeseq2 handoff.
• references/single_cell_alevin.md — why salmon alevin is removed and the piscem + alevin-fry replacement.

Part of the AlterLab Academic Skills suite.