Enhanced detection of RNA modifications and read mapping with high-accuracy nanopore RNA basecalling models

IF 6.2 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Genome research Pub Date : 2024-09-13 DOI:10.1101/gr.278849.123
Gregor Diensthuber, Leszek P Pryszcz, Laia Llovera, Morghan C Lucas, Anna Delgado-Tejedor, Sonia Cruciani, Jean-Yves Roignant, Oguzhan Begik, Eva Maria Novoa
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Abstract

In recent years, nanopore direct RNA sequencing (DRS) became a valuable tool for studying the epitranscriptome, due to its ability to detect multiple modifications within the same full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling 'errors' in DRS datasets, N6-methyladenosine (m6A) modifications produce relatively low 'errors' compared to other RNA modifications, limiting the applicability of this approach to m6A sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully unmodified sequences, increases the 'error'signal of m6A, leading to enhanced detection and improved sensitivity even at low stoichiometries. Moreover, we find that high-accuracy alternative RNA basecalling models can show up to 97% median basecalling accuracy, outperforming currently available RNA basecalling models, which show 91% median basecalling accuracy. Notably, the use of high-accuracy basecalling models is accompanied by a significant increase in the number of mapped reads –especially in shorter RNA fractions– and increased basecalling error signatures at pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) modified sites. Overall, our work demonstrates that alternative RNA basecalling models can be used to improve the detection of RNA modifications, read mappability, and basecalling accuracy in nanopore DRS datasets.
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利用高精度纳米孔 RNA 基调用模型增强 RNA 修饰检测和读图能力
近年来,纳米孔直接 RNA 测序(DRS)因其能够检测同一全长原生 RNA 分子中的多种修饰而成为研究表转录组的重要工具。在 DRS 数据集中,RNA 修饰可以以系统性基调 "误差 "的形式被识别出来,但与其他 RNA 修饰相比,N6-甲基腺苷(m6A)修饰产生的 "误差 "相对较低,从而限制了这种方法对高化学计量比修饰的 m6A 位点的适用性。在这里,我们证明了使用完全未修饰序列训练的替代 RNA 基信号模型会增加 m6A 的 "误差 "信号,从而提高检测能力和灵敏度,即使在低化学计量比的情况下也是如此。此外,我们发现高精度替代 RNA 基调模型的中位基调准确率可达 97%,优于目前可用的 RNA 基调模型,后者的中位基调准确率为 91%。值得注意的是,在使用高精度基调模型的同时,映射读数的数量也显著增加--尤其是在较短的 RNA 片段中--并且假尿嘧啶(Ψ)和 N1-甲基假尿嘧啶(m1Ψ)修饰位点的基调错误特征也增加了。总之,我们的工作表明,替代的 RNA 基调模型可用于改进纳米孔 DRS 数据集中 RNA 修饰的检测、读取映射性和基调准确性。
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来源期刊
Genome research
Genome research 生物-生化与分子生物学
CiteScore
12.40
自引率
1.40%
发文量
140
审稿时长
6 months
期刊介绍: Launched in 1995, Genome Research is an international, continuously published, peer-reviewed journal that focuses on research that provides novel insights into the genome biology of all organisms, including advances in genomic medicine. Among the topics considered by the journal are genome structure and function, comparative genomics, molecular evolution, genome-scale quantitative and population genetics, proteomics, epigenomics, and systems biology. The journal also features exciting gene discoveries and reports of cutting-edge computational biology and high-throughput methodologies. New data in these areas are published as research papers, or methods and resource reports that provide novel information on technologies or tools that will be of interest to a broad readership. Complete data sets are presented electronically on the journal''s web site where appropriate. The journal also provides Reviews, Perspectives, and Insight/Outlook articles, which present commentary on the latest advances published both here and elsewhere, placing such progress in its broader biological context.
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