{
  "id": 13391728,
  "title": "RiboFlow v2: a configurable ribosome profiling pipeline reveals measurements sensitive to read-mapping methodology",
  "url": "https://urgent.news/2026/10/10/riboflow-v2-a-configurable-ribosome-profiling-pipeline-reveals",
  "topic": "science",
  "section": "Science",
  "published": "2026-10-10T00:00:00.000Z",
  "source": {
    "name": "bioRxiv",
    "slug": "biorxiv",
    "url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756038v1?rss=1"
  },
  "original_language": "en",
  "account": "RiboFlow v2 is a versatile ribosome profiling pipeline that scrutinizes measurements susceptible to read-mapping methodology. Ribosome profiling experiments involve sequencing ribosome-protected fragments (RPFs) to capture a momentary translation view. Alignment to a well-curated transcriptome usually captures most RPF signal, but can overlook ribosome activity when expressed exons are absent from the reference. Conversely, genome alignment of RPFs broadens the detectable sequence space but can introduce ambiguity between homologous loci. Existing workflows often pair reference choice with handling ambiguously mapped reads, making it challenging to separate the impacts of either decision on downstream analyses.\n\nTo address this, the authors developed RiboFlow v2, which augments the existing transcriptome workflow with genome alignment and configurable handling of multimapping reads. By applying RiboFlow v2 to 24 human ribosome profiling libraries alongside their matching RNA-seq data, the researchers discovered that nucleotide-level coverage at matched coding-sequence positions and translation efficiency (TE) were generally consistent between the alignment strategies when strict alignment filters were applied. Despite these conditions, individual genes exhibited significant variations in coverage and TE estimates. Some of these discrepancies stemmed from coverage in alternative exons not included in selected transcripts and ambiguous alignments occurring between protein-coding genes and processed pseudogenes.\n\nEmploying clustering techniques based on read-assignment patterns, the authors identified groups of genes linked to pseudogene prevalence. Moreover, they found that shared exonic sequence among reference transcripts contributes to alignment discrepancies. RiboFlow v2 surpasses the analysis of selected reference transcripts, enabling the identification of genes whose ribosome coverage and abundance estimates hinge on reference composition and the handling of ambiguously mapped reads.",
  "summary": "Ribosome profiling experiments capture a snapshot of translation by sequencing ribosome-protected fragments (RPFs). Alignment to a well-curated transcriptome captures most RPF signal, but can miss ribosome activity when expressed exons are absent from the reference. Genome alignment of RPFs expands the detectable sequence space, but can expose ambiguity between homologous loci. Existing workflows…",
  "key_points": [],
  "editors_take": null,
  "illustration": null,
  "coverage": {
    "outlets": 1,
    "also_reported_by": []
  },
  "ai_generated": true,
  "disclaimer": "Summaries, key points and the editor’s take are written by software from other outlets’ reporting and may contain errors — always check the linked original."
}