{
  "id": 4335156,
  "title": "Ultra-High Multiplexing Enables Near-Full-Length 16S rRNA Gene Amplicon Sequencing of Over 1,200 Gut Microbiome Samples on a Single Nanopore Flow Cell",
  "url": "https://urgent.news/2026/08/29/ultra-high-multiplexing-enables-near-full-length-16s-rrna-gene",
  "topic": "science",
  "section": "Science",
  "published": "2026-08-29T00:00:00.000Z",
  "source": {
    "name": "bioRxiv",
    "slug": "biorxiv",
    "url": "https://www.biorxiv.org/content/10.64898/2026.08.29.747698v1?rss=1"
  },
  "original_language": "en",
  "account": "Recent advancements in Oxford Nanopore Technologies have pushed the boundaries of 16S rRNA gene analysis in gut microbiome research. The company's R10.4.1 flow cell chemistry has significantly enhanced sequencing accuracy, overcoming the short read length limitations of traditional platforms like Illumina. By employing a custom multiple-primer strategy, researchers can generate near-full-length amplicons, facilitating read-by-read taxonomic classification - a feat previously unattainable with short-read sequencing technologies. While the multiple-primer approach could theoretically handle sequencing of over 18,000 samples (192 x 96), the current flow cell capacity proves sufficient for analyzing approximately 1,000-1,500 samples. To verify the scalability and effectiveness of their per-read classification pipeline, the researchers spiked human fecal microbiome samples with two additional bacterial strains (Imtechella halotolerans and Allobacillus halotolerans) not typically found in human fecal samples. Strikingly, more than a thousand samples could be successfully sequenced on a single flow cell, achieving an exceptionally low per-molecule error rate necessary for direct per-read classification. Moreover, the sequencing depth was adequate for downstream analysis. This breakthrough in scalability dramatically reduces per-sample costs, making ultra-high multiplexing a more accessible option for a wider range of researchers. To capitalize on these advancements, the team developed RubyRed, a processing pipeline that handles raw sequencing data and assigns taxonomic classifications on a per-read basis. The researchers validated RubyRed's performance using spike-in references (I. halotolerans and A. halotolerans), achieving impressive mean single-read sequencing accuracy of 99% and 98.9%, respectively. The majority of reads also surpassed the canonical threshold required for species-level taxonomic classification based on the 16S rRNA gene.",
  "summary": "Next-generation sequencing (NGS) of the prokaryotic 16S rRNA gene revolutionized gut microbiome research two decades ago. However, short read lengths remain an inherent limitation of platforms such as the widely used Illumina platforms (2 x 150-300 bp). Recent advances in Oxford Nanopore Technologies (ONT) flow cell chemistry (R10.4.1) have substantially improved sequencing accuracy. Combined…",
  "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."
}