bioRxivMolecular BiologyPreprint
SIRVs: Spike-In RNA Variants as External Isoform Controls in RNA-Sequencing
Lukas PauliD, Petra Kubala, Gudrun Horner, Michael AnteiD, Igor Holländer, Alexander SeitziD, Torsten Reda
Abstract
Spike-In RNA variants (SIRVs) enable for the first time the validation of RNA sequencing workflows using external isoform transcript controls. 69 transcripts, derived from seven human model genes, cover the eukaryotic transcriptome complexity of start- and end-site variations, alternative splicing, overlapping genes, and antisense transcription in a condensed format. Reference RNA samples were spiked with SIRV mixes, sequenced, and exemplarily four data evaluation pipelines were challenged to account for biases introduced by the RNA-Seq workflow. The deviations of the respective isoform quantifications from the known inputs allow to determine the comparability of sequencing experiments and to extrapolate to which degree alterations in an RNA-Seq workflow affect gene expression measurements. The SIRVs as external isoform controls are an important gauge for inter-experimental comparability and a modular spike-in contribution to clear the way for diagnostic RNA-Seq applications.
- organism: human
- assay: bulk RNA-seq
- research focus: benchmarking
- research focus: differential expression
- research focus: variant calling
- approach: statistical model
Abstract summary
Claude Opus 5.5 · xhigh effort · 2 Nov 2026 · from the abstract
Spike-In RNA Variants (SIRVs) provide 69 external isoform control transcripts derived from seven human model genes to benchmark isoform quantification accuracy and workflow bias across RNA-Seq pipelines.
Validating how RNA-Seq workflow alterations and evaluation pipelines affect isoform-level expression measurements requires external controls that capture eukaryotic transcript complexity. Spike-In RNA Variants (SIRVs) introduce 69 external isoform controls derived from seven human model genes covering alternative splicing, start- and end-site variation, overlapping genes, and antisense transcription.
Key results
ShownA 69-transcript Spike-In RNA Variant (SIRV) set derived from seven human model genes captures eukaryotic isoform complexity in a condensed external control panel.
Spans antisense transcription, overlapping genes, alternative splicing, and variations in transcript start and end sites across 69 transcripts from seven human model genes. · Abstract
ShownSpiking SIRV mixes into reference RNA samples and challenging four data evaluation pipelines measures deviations between quantified isoforms and known inputs.
Evaluated across spiked reference RNA samples and four RNA-Seq data evaluation pipelines to assess workflow-induced biases and inter-experimental comparability. · Abstract
Methods, data and reproducibility
Designed 69 Spike-In RNA Variant (SIRV) transcripts from seven human model genes representing diverse transcript structures, spiked SIRV mixes into reference RNA samples for RNA sequencing, and evaluated isoform quantification deviations across four data evaluation pipelines.
- Reference RNA samples spiked with 69 Spike-In RNA Variant (SIRV) transcripts derived from seven human model genes · human · n = 69 transcripts from 7 human model genes
- Methods & tools
- Spike-In RNA Variants (SIRVs)
- Reproducibility gaps
- Abstract only; the names of the four evaluation pipelines, sequencing accessions, and quantitative deviation metrics are not stated in the abstract.
Limitations (1)
- The abstract does not identify the four evaluated data analysis pipelines or report numerical isoform quantification deviations. · noted in this summary
Read this if you benchmark isoform quantification pipelines or need external spike-in controls to measure inter-experimental comparability in RNA-Seq workflows.
- Citations
- 46
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