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A comparative evaluation of computational models for RNA modification detection using nanopore sequencing with RNA004 chemistry.

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  • Additional Information
    • Source:
      Publisher: Oxford University Press Country of Publication: England NLM ID: 100912837 Publication Model: Print Cited Medium: Internet ISSN: 1477-4054 (Electronic) Linking ISSN: 14675463 NLM ISO Abbreviation: Brief Bioinform Subsets: MEDLINE
    • Publication Information:
      Publication: Oxford : Oxford University Press
      Original Publication: London ; Birmingham, AL : H. Stewart Publications, [2000-
    • Subject Terms:
      Nanopore Sequencing*/methods ; RNA*/genetics ; RNA*/chemistry ; Sequence Analysis, RNA*/methods; Computational Biology/methods ; Adenosine/analogs & derivatives ; Humans ; HEK293 Cells ; HeLa Cells
    • Abstract:
      Direct RNA sequencing from Oxford Nanopore Technologies has become a valuable method for studying RNA modifications such as N6-methyladenosine (m6A) and pseudouridine (pseU). Recent advancements in the RNA004 chemistry substantially reduce sequencing errors compared to previous chemistries, promising enhanced accuracy for epitranscriptomic analysis. Here we benchmark the performance of two RNA modification detection models for RNA004 data, Dorado and m6Anet, using two wild-type (WT) cell lines (HEK293T and HeLa), with respective ground truths from GLORI and eTAM-seq, and in vitro transcribed (IVT) RNA as negative controls. We found that for m6A sites with ≥10% modification ratio and ≥ 10X coverage, Dorado has higher recall (~0.92) than m6Anet (~0.51). Among true positive predictions, there are high correlations of m6A modification stoichiometry (correlation coefficient of ~0.89 for Dorado-truth and ~ 0.72 for m6Anet-truth). However, combined assessment of WT and IVT datasets show that while the per-site false positive rate can be lower (~8% for Dorado and ~ 33% for m6Anet), both tools can have high per-site false discovery rate of m6A (~40% for Dorado and ~ 80% for m6Anet), or for pseU (~95% for Dorado). Motif analysis reveals that both tools exhibit high heterogeneity of false positive calls across sequence contexts. There is also a substantial overlap of false positive calls between the two IVT samples, suggesting a filtering strategy by compiling a set of low-confidence sites from diverse IVT samples. Our analysis highlights key strengths and limitations of the current generation of m6A detection algorithms and offers insights into optimizing thresholds and interpretability.
      (© The Author(s) 2025. Published by Oxford University Press.)
    • Comments:
      Update of: bioRxiv. 2025 Feb 08:2025.02.03.636352. doi: 10.1101/2025.02.03.636352.. (PMID: 39975272)
      Erratum in: Brief Bioinform. 2025 Aug 31;26(5):bbaf588. doi: 10.1093/bib/bbaf588.. (PMID: 41140068)
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    • Grant Information:
      R01 HG013359 United States HG NHGRI NIH HHS; U01 CA279618 United States CA NCI NIH HHS; Children's Hospital of Philadelphia Research Institute; HG013359 to K.W., CA279618 to Y.H. United States NH NIH HHS
    • Contributed Indexing:
      Keywords: in vitro transcription; N6-methyladenosine; Oxford Nanopore sequencing; RNA modifications; benchmarking; pseudouridine
    • Accession Number:
      63231-63-0 (RNA)
      CLE6G00625 (N-methyladenosine)
      K72T3FS567 (Adenosine)
    • Publication Date:
      Date Created: 20250813 Date Completed: 20250826 Latest Revision: 20260127
    • Publication Date:
      20260130
    • Accession Number:
      PMC12346441
    • Accession Number:
      10.1093/bib/bbaf404
    • Accession Number:
      40802798