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.

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 --gcBias." / "Use salmon's --sketch mode."
• "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

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.

Two Critical Correctness Traps (read before quantifying)

These are the three failures most outdated RNA-seq instructions get wrong now that salmon has moved to 2.x (a from-scratch Rust rewrite; bioconda 2.7.0). All are confirmed in the upstream MIGRATION.md (see references/tool_versions.md).

1. salmon 2.0 cannot read a C++ (pufferfish) index. The index format changed

with the rewrite. Loading an old index — or pointing old salmon at a 2.x index — is detected and rejected with a clear error, so this fails loudly rather than silently. Rebuild every index with the same salmon version you quantify with. quant.sf and the bootstrap/Gibbs outputs are unchanged, so tximport, tximeta, fishpond and swish keep working on 2.x output with no changes.

1. `--validateMappings` no longer does anything. Selective alignment is the

default (and only) alignment mode in 2.x, so the flag parses and logs a warning. Copying it from a 2019-era tutorial is harmless but misleading; drop it. Several other C++ flags now error out: --mimicBT2, --mimicStrictBT2, --minAssignedFrags, --numBiasSamples, --alternativeInitMode.

1. `salmon alevin` was REMOVED. Single-cell quantification is no longer part of

salmon; salmon alevin … prints a redirect and exits. Use the piscem + alevin-fry ecosystem instead. If the user has single-cell / droplet data, route per the table above and see references/single_cell_alevin.md.

If you genuinely need the old behavior, the final C++ release (salmon 1.12.0) lives on the upstream cpp branch and is packaged separately as salmon-cpp.

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 under salmon 2.x — 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 \
  --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.

• Selective alignment is the default in salmon 2.x — there is no flag to turn

it on (--validateMappings is accepted and ignored). --sketch opts out of it into faster alignment-free pseudoalignment; prefer the default when the quantification feeds differential expression, and reserve --sketch for very large screens where speed dominates.

• `--gcBias` — corrects fragment-level GC bias; recommended for DE and cheap

to enable. Add --seqBias for 5'/3' sequence-specific bias if needed.

• `--ignoreTxVersion` (new in 2.x) — with -g/--geneMap, matches transcript

IDs ignoring the trailing .N, the way tximport's option of the same name does. Needed for an Ensembl cDNA index against an Ensembl GTF. When transcripts fail to match the gene map, 2.x writes the offending names to aux_info/genemap_unmatched_txps.json and warns once with a count, instead of C++ salmon's one warning per transcript — check for that file after any run that used -g.

• 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).

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.

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.

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.

Self-Check Before Reporting

• Did you rebuild the salmon index with the same 2.x binary you quantified with

(trap #1)? A C++/pufferfish index is rejected outright.

• 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?

References

• references/tool_versions.md — pinned versions

(salmon 2.x, kallisto v0.52.0, kb-python, nf-core/rnaseq) and the upstream release-note facts (the 2.0 rewrite and index break, 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.