Evaluation and Application of Tools for mRNA Isoform Detection Using Nanopore Sequencing Data

Abstract

Alternative splicing contributes to RNA isoform diversity, enhancing functional complexity in biological systems. Recent advancements in long-read sequencing technologies, notably those by Oxford Nanopore Technologies (ONT), facilitate comprehensive analysis of RNA isoforms through the generation of longer reads capable of capturing full-length transcripts. However, such advancements introduce challenges associated with complex long-read data, particularly in terms of accurate basecalling and robust isoform identification. In this study, we benchmarked two basecalling algorithms, Guppy and Dorado, utilizing real ONT sequencing data, finding that Dorado greatly enhanced read length and quality, thus improving downstream isoform analysis accuracy. Subsequently, we evaluated five isoform identification and quantification tools (Bambu, IsoQuant, FLAIR, StringTie2, and lr-kallisto) using spike-in RNA datasets as a ground truth. To further dissect how sequencing parameters (read length, read accuracy, sequencing depth, and transcript length) impact tool performance, we generated 96 synthetic ONT RNA datasets. We identified distinct strengths and weaknesses for each tool in isoform discovery depending on varying sequencing qualities and provided guidance for selecting the most suitable tool based on specific research questions. Among the evaluated tools, Bambu exhibited superior performance under the specific conditions of our datasets. Applying the optimized workflow, we compared transcriptional profiles of lung and bladder epithelial cells following exposure to IFN-β, an antiviral cytokine. Notably, we discovered that a truncated form of RIG-I, a key receptor detecting dsRNA viruses such as influenza and coronaviruses, is specifically expressed in lung epithelial cells but not in bladder epithelial cells in response to IFN-β. These findings underscore the critical importance of selecting appropriate basecalling algorithms and isoform identification tools tailored to the characteristics of the sequencing data, ultimately enabling the identification of cell-type-specific isoform variations with potential therapeutic implications for viral infections.