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<p>A-to-I RNA editing, one of the most frequent RNA modifications, is essential for most aspects of RNA metabolism. However, the accuracy of quantifying A-to-I editing levels has been largely neglected, particularly when contrasting next-generation sequencing (NGS) with long-read sequencing (LRS) RNA-seq. To address it, we performed paired NGS and LRS cDNA RNA-seq of both HEK293T and U2OS cells, revealing that the A-to-I editing levels were estimated to be lower by NGS compared with LRS, a conclusion that was confirmed through full-length amplicon sequencing. Furthermore, the lower estimation of A-to-I editing levels is widespread, while we also identified this consequence using released public both same-study and cross-study NGS and LRS RNA-seq data in various human cancer cell lines. In addition, our analyses indicate that the primary factor contributing to this lower estimation is the sequencing read length difference, and a significantly positive correlation between the quantification and read length is identified by analyzing the public NGS RNA-seq data of various cell lines. We additionally found that shorter sequencing reads are more prone to aligning incorrectly to the reference genome, such as uniquely-to-multiply mapped and uniquely mapped-to-unmapped, inducing the lower estimation of A-to-I editing levels. In summary, our analyses indicate that LRS RNA-seq is preferable for accurately quantifying A-to-I editing levels.</p>

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Short-read RNA-seq yields lower estimates of A-to-I RNA editing levels than long-read cDNA sequencing

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月22日

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  • Shi Cheng,
  • Yunling Qi,
  • Jiaqi Ya,
  • Lei Xia,
  • Wenshan Zhang,
  • Qing Xiong,
  • Qiuli Liu,
  • Jiaxing Zhang,
  • Yulong Song

摘要

A-to-I RNA editing, one of the most frequent RNA modifications, is essential for most aspects of RNA metabolism. However, the accuracy of quantifying A-to-I editing levels has been largely neglected, particularly when contrasting next-generation sequencing (NGS) with long-read sequencing (LRS) RNA-seq. To address it, we performed paired NGS and LRS cDNA RNA-seq of both HEK293T and U2OS cells, revealing that the A-to-I editing levels were estimated to be lower by NGS compared with LRS, a conclusion that was confirmed through full-length amplicon sequencing. Furthermore, the lower estimation of A-to-I editing levels is widespread, while we also identified this consequence using released public both same-study and cross-study NGS and LRS RNA-seq data in various human cancer cell lines. In addition, our analyses indicate that the primary factor contributing to this lower estimation is the sequencing read length difference, and a significantly positive correlation between the quantification and read length is identified by analyzing the public NGS RNA-seq data of various cell lines. We additionally found that shorter sequencing reads are more prone to aligning incorrectly to the reference genome, such as uniquely-to-multiply mapped and uniquely mapped-to-unmapped, inducing the lower estimation of A-to-I editing levels. In summary, our analyses indicate that LRS RNA-seq is preferable for accurately quantifying A-to-I editing levels.