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<p>Long non-coding RNAs (lncRNAs) play crucial roles in gene regulation, but their full-length isoforms are often missed because of the limitations of poly(A)-based enrichment and short-read sequencing. Here, we aim to establish a comprehensive transcriptome profiling that captures both poly(A)+ and poly(A)- RNA isoforms using Oxford Nanopore Technologies (ONT) R10.4.1 flowcells. We establish an rRNA-depleted full-length transcriptome sequencing workflow (NanoncRNA-Seq), and use it together with Illumina NovaSeq to profile lncRNA isoforms in <i>Saccharomyces cerevisiae</i> under glucose and ethanol-associated physiological states. We combine multiple analytical tools to evaluate expression levels, splicing patterns, variants, and lncRNA identification. ONT sequencing achieves high accuracy (Q-score: 22.35, 99.42%) and detects fewer SNP and more novel isoforms, while Illumina sequencing reports fewer INDELs. Expression profiles are highly consistent within each platform and moderately across platforms. Notably, NanoncRNA-seq enables isoform-resolved lncRNA discovery and recoveres substantially more lncRNAs than Illumina (Pinfish: <i>n</i> = 260; Illumina: <i>n</i> = 51), including more lincRNAs (<i>n</i> = 201 vs. <i>n</i> = 25), likely because low-abundance transcripts are difficult to reconstruct from short reads. Overall, NanoncRNA-Seq effectively captures full-length lncRNA isoform discovery and highlights the complementary strengths of ONT in transcriptome research.</p><p></p>

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An rRNA-depleted full-length transcriptome strategy using nanopore sequencing for identification of novel lncRNA isoforms

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月27日

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  • Tianyuan Zhang,
  • Jie Chen,
  • Huiyu Hou,
  • Salsabeel Yousuf,
  • Jinlong Ji,
  • Yali Li,
  • Zhaoyang Tian,
  • Dengli Guo,
  • Huanyu Tang,
  • Ting Qiu,
  • Xiaojing Li,
  • Weike Zeng,
  • Man Lu,
  • Yao Chen,
  • Liu Yang,
  • Hu Chen,
  • Zhipeng Qu,
  • Kai Wu,
  • Tingyu Ma,
  • Yong-Xin Liu

摘要

Long non-coding RNAs (lncRNAs) play crucial roles in gene regulation, but their full-length isoforms are often missed because of the limitations of poly(A)-based enrichment and short-read sequencing. Here, we aim to establish a comprehensive transcriptome profiling that captures both poly(A)+ and poly(A)- RNA isoforms using Oxford Nanopore Technologies (ONT) R10.4.1 flowcells. We establish an rRNA-depleted full-length transcriptome sequencing workflow (NanoncRNA-Seq), and use it together with Illumina NovaSeq to profile lncRNA isoforms in Saccharomyces cerevisiae under glucose and ethanol-associated physiological states. We combine multiple analytical tools to evaluate expression levels, splicing patterns, variants, and lncRNA identification. ONT sequencing achieves high accuracy (Q-score: 22.35, 99.42%) and detects fewer SNP and more novel isoforms, while Illumina sequencing reports fewer INDELs. Expression profiles are highly consistent within each platform and moderately across platforms. Notably, NanoncRNA-seq enables isoform-resolved lncRNA discovery and recoveres substantially more lncRNAs than Illumina (Pinfish: n = 260; Illumina: n = 51), including more lincRNAs (n = 201 vs. n = 25), likely because low-abundance transcripts are difficult to reconstruct from short reads. Overall, NanoncRNA-Seq effectively captures full-length lncRNA isoform discovery and highlights the complementary strengths of ONT in transcriptome research.