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Biotechnol."],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats: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.<\/jats:p>","DOI":"10.1007\/s44307-026-00123-w","type":"journal-article","created":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T03:31:13Z","timestamp":1784691073000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Short-read RNA-seq yields lower estimates of A-to-I RNA editing levels than long-read cDNA sequencing"],"prefix":"10.1007","volume":"4","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0092-564X","authenticated-orcid":false,"given":"Shi","family":"Cheng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunling","family":"Qi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaqi","family":"Ya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Xia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenshan","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing","family":"Xiong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiuli","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxing","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yulong","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,7,22]]},"reference":[{"key":"123_CR1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.0020391","volume":"2","author":"A Athanasiadis","year":"2004","unstructured":"Athanasiadis A, Rich A, Maas S. 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