{
  "abstract": "Background: Next-generation sequencing (NGS) is vital for tracking viral evolution during outbreaks. Oxford Nanopore Technologies (ONT) offers long-read, cost-effective sequencing, but accurate sub-consensus variant detection remains challenging. LoFreq, a widely used caller for sub-consensus variant calling in haploid genomes, was designed for short reads. This study assesses and benchmarks LoFreq variant identification accuracy on long-read data and proposes a calibration method to improve accuracy. Methods: We created truth sets using three SARS-CoV-2 spike gene plasmids (7179 bases, 100 SNVs) and whole Escherichia coli genomes. Libraries were sequenced using R9.4.1 and R10.4.1 flow cells. LoFreq’s recall was benchmarked across chemistries and library sizes. We also introduced a method to adjust Phred quality scores for enhanced accuracy. Results: LoFreq showed high sensitivity, detecting variants at frequencies as low as 0.1%, with best performance on R10.4.1. However, false discovery rates (FDR) were chemistry- and depth-dependent. Our Phred score calibration reduced false positives while maintaining recall, though it was less effective below 10% frequency and not suitable for structural variant detection. Conclusion: LoFreq can detect sub-consensus variants in ONT data, but high FDR limits direct use. Our calibration method improves accuracy, offering a simple Phred score adjustment approach that reduces false discovery rates in ONT data.",
  "abstract_shown": true,
  "affiliations": [
    "National University of Singapore, Saw Swee hock School of Public Health, Singapore, Singapore",
    "ImmunoScape, Pte Ltd, Singapore, Singapore",
    "Oxford Nanopore Technologies, Oxford, UK",
    "Department of Pharmacy, National University of Singapore, Singapore, Singapore"
  ],
  "article_type": "Research Article",
  "arxiv": null,
  "author_total": 9,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Xavier Zair"
    },
    {
      "affs": [
        1
      ],
      "name": "Andreas Wilm"
    },
    {
      "affs": [
        2
      ],
      "name": "Miles C. Benton"
    },
    {
      "affs": [
        3
      ],
      "name": "Cheng Yong Tham"
    },
    {
      "name": "Lin Yang"
    },
    {
      "name": "Paola Florez de Sessions"
    },
    {
      "name": "October Michael Sessions"
    },
    {
      "name": "Eng Hui Chew"
    },
    {
      "name": "Swapnil Mishra"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/acsnnq0pkfnd2.bib",
  "cited_by": 0,
  "doi": "10.1371/journal.pone.0356259",
  "fulltext_url": null,
  "id": "acsnnq0pkfnd2",
  "issns": [
    "1932-6203"
  ],
  "landing_url": "https://doi.org/10.1371/journal.pone.0356259",
  "language": null,
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
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      "openalex",
      "1312|Genomics and Phylogenetic Studies"
    ],
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      "plos",
      "/Biology and life sciences/Computational biology/Genome analysis"
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      "/Biology and life sciences/Computational biology/Genome analysis/Transcriptome analysis/Next-generation sequencing"
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      "/Biology and life sciences/Genetics/Genetic loci/Alleles"
    ],
    [
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      "/Biology and life sciences/Genetics/Genomics"
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    [
      "plos",
      "/Biology and life sciences/Genetics/Genomics/Genome analysis"
    ],
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      "plos",
      "/Biology and life sciences/Genetics/Genomics/Genome analysis/Transcriptome analysis/Next-generation sequencing"
    ],
    [
      "plos",
      "/Biology and life sciences/Genetics/Genomics/Microbial genomics/Bacterial genomics"
    ],
    [
      "plos",
      "/Biology and life sciences/Genetics/Microbial genetics/Bacterial genetics/Bacterial genomics"
    ],
    [
      "plos",
      "/Biology and life sciences/Microbiology/Bacteriology/Bacterial biochemistry"
    ],
    [
      "plos",
      "/Biology and life sciences/Microbiology/Bacteriology/Bacterial genetics/Bacterial genomics"
    ],
    [
      "plos",
      "/Biology and life sciences/Microbiology/Medical microbiology/Microbial pathogens/Bacterial pathogens/Escherichia coli"
    ],
    [
      "plos",
      "/Biology and life sciences/Microbiology/Microbial genomics/Bacterial genomics"
    ],
    [
      "plos",
      "/Biology and life sciences/Molecular biology/Molecular biology techniques/Sequencing techniques/DNA sequencing/Next-generation sequencing"
    ],
    [
      "plos",
      "/Biology and life sciences/Organisms/Bacteria/Enterobacteriaceae/Escherichia/Escherichia coli"
    ],
    [
      "plos",
      "/Biology and life sciences/Organisms/Bacteria/Gut bacteria/Escherichia/Escherichia coli"
    ],
    [
      "plos",
      "/Medicine and health sciences/Pathology and laboratory medicine/Pathogens/Microbial pathogens/Bacterial pathogens/Escherichia coli"
    ],
    [
      "plos",
      "/Research and analysis methods/Animal studies/Experimental organism systems/Model organisms/Escherichia coli"
    ],
    [
      "plos",
      "/Research and analysis methods/Animal studies/Experimental organism systems/Prokaryotic models/Escherichia coli"
    ],
    [
      "plos",
      "/Research and analysis methods/Database and informatics methods/Bioinformatics/Sequence analysis/Sequence alignment"
    ],
    [
      "plos",
      "/Research and analysis methods/Model organisms/Escherichia coli"
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    [
      "plos",
      "/Research and analysis methods/Molecular biology techniques/Sequencing techniques/DNA sequencing/Next-generation sequencing"
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  "pdf_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0356259&type=printable",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-05",
  "publisher": "Public Library of Science",
  "ris": "https://research.draconitic.com/paper/acsnnq0pkfnd2.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:37Z",
      "native_id": "10.1371/journal.pone.0356259",
      "source": "plos",
      "url": "https://doi.org/10.1371/journal.pone.0356259"
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  "subjects": [
    {
      "field": "biology",
      "origin": "plos:computational biology",
      "subfield": "bioinformatics"
    },
    {
      "field": "biology",
      "origin": "plos:genetics",
      "subfield": "genetics-genomics"
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    {
      "field": "biology",
      "origin": "plos:microbiology",
      "subfield": "microbiology"
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  "title": "Sub-consensus haploid variant calling in long-read sequencing technology",
  "title_text": "Sub-consensus haploid variant calling in long-read sequencing technology",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:17:37Z",
  "url": "https://research.draconitic.com/paper/acsnnq0pkfnd2",
  "venue": "PLOS One",
  "versions": []
}