Oarfish Quantification Format Handling

oarfish-quantification-format-handling

Summary

Import and extract transcript-level abundance, count, and length matrices from oarfish long-read RNA-seq quantification files (quant.gz format) using tximport. This skill is essential when working with oarfish-quantified long-read data that must be integrated into downstream differential expression workflows.

When to use

You have long-read RNA-seq samples quantified by oarfish (output as quant.gz files) and need to extract transcript-level or gene-level abundance, count, and length matrices for downstream statistical analysis. Use this skill when you want to preserve transcript-level information during import rather than immediately aggregating to gene level, or when you need length matrices to account for differential isoform usage across samples.

When NOT to use

• Input files are already in gene-level count matrix format (use directly with DESeq2 or edgeR instead of re-importing)
• Quantification was performed with a different tool (salmon, sailfish, kallisto); use the appropriate type parameter instead
• You require inferential replicates (Gibbs or bootstrap samples); verify oarfish output includes these before attempting import

Inputs

• oarfish quant.gz files (one per sample)
• sample metadata or design table (recommended for downstream use)
• tx2gene mapping data.frame (required if gene-level summarization is desired)

Outputs

• abundance matrix (transcript or gene level, rows = features, columns = samples)
• counts matrix (estimated counts, same dimensions as abundance)
• length matrix (average transcript length weighted by sample-specific abundance)

How to apply

Load oarfish quant.gz files using tximport with the type='oarfish' parameter to specify the quantification software. Set txOut=TRUE to retain transcript-level output matrices instead of gene-level summarization; omit or set txOut=FALSE if gene-level aggregation is desired. Extract the returned list object containing three matrices: 'abundance' (normalized transcript abundances), 'counts' (estimated transcript counts), and 'length' (transcript lengths weighted by sample-specific abundances). The length matrix can subsequently be used as an offset for downstream gene-level differential analysis to correct for potential changes in gene length arising from differential isoform usage. Verify that matrix dimensions match the number of samples and transcripts/genes, and check for consistency of row names (transcript or gene IDs) and column names (sample identifiers) across replicates.

Related tools

• tximport (Primary import function; accepts type='oarfish' parameter and txOut flag to control output level and extract matrices) — github:thelovelab__tximport
• DESeq2 (Downstream differential expression analysis using imported count matrices and length-based offsets)
• edgeR (Alternative downstream differential expression analysis using imported count matrices and length-based offsets)
• limma-voom (Alternative downstream differential expression analysis using imported count matrices and length-based offsets)

Examples

txi <- tximport(files = c('sample1.quant.gz', 'sample2.quant.gz', 'sample3.quant.gz'), type = 'oarfish', txOut = TRUE)

Evaluation signals

• Returned abundance, counts, and length matrices have consistent dimensions (same number of rows = features, same number of columns = samples)
• Row names match expected transcript or gene IDs from the quantification; column names match input sample identifiers
• Abundance values are in expected range (typically 0–1 or log-scale); counts are non-negative integers or near-integer estimates; length values are positive
• No missing (NA) or infinite values in returned matrices; all samples are represented
• Downstream differential analysis (DESeq2/edgeR/limma-voom) accepts the matrices without format errors and length offset can be incorporated

Limitations

• tximport requires a tx2gene mapping only if gene-level summarization is desired; transcript-level output (txOut=TRUE) does not require this mapping
• The length matrix represents average transcript length weighted by abundance and may vary across samples due to differential isoform usage; this is intentional and supports correction for composition bias in downstream analysis
• No automatic metadata annotation is provided by tximport itself; for automatic addition of annotation metadata, use the tximeta package instead

Evidence

• [other] transcript-level import with txOut parameter: "tximport with type='oarfish' and txOut=TRUE successfully imports transcript-level abundance, count, and length matrices from oarfish quant.gz files"
• [intro] length matrix for offset calculation: "Average transcript length, weighted by sample-specific transcript abundance estimates, is provided as a matrix which can be used as an offset for different expression of gene-level counts"
• [readme] tximport function purpose: "Imports transcript-level abundance, estimated counts and transcript lengths, and summarizes into matrices for use with downstream statistical analysis packages such as edgeR, DESeq2, limma-voom"
• [intro] type parameter specification: "The tximport package has a single function for importing transcript-level estimates. The type argument is used to specify what software was used for estimation"
• [intro] correction for differential isoform usage: "this approach corrects for potential changes in gene length across samples (e.g. from differential isoform usage)"