RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community’s ability to interrogate both forms of RNA variation simultaneously. We employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis. Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

中文翻译：

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使用 FLAIR2 检测长读段中单倍型特异性转录本变异


RNA-seq 在 RNA 加工异常方面取得了重大发现，表明这些 RNA 变异与多种疾病有关。 RNA 中的异常剪接和单核苷酸变异 (SNV) 已被证明会改变转录物稳定性、定位和功能。特别是，ADAR（一种介导腺苷至肌苷编辑的酶）的上调先前已被认为与肺腺癌细胞侵袭性的增加以及剪接调节有关。尽管研究剪接和 SNV 具有重要的功能，但短读长 RNA-seq 的使用限制了社区同时研究两种形式的 RNA 变异的能力。我们采用长读长测序技术来获得全长转录本序列，在单分子水平上阐明变体对剪接变化的顺式效应。我们开发了一个增强 FLAIR 的计算工作流程，FLAIR 是一种调用长读数据中表达的同工型模型的工具，以将 RNA 变体调用与承载它们的相关同工型整合起来。我们从敲低和未敲低 ADAR 的 H1975 肺腺癌细胞中生成具有高序列准确性的纳米孔数据。我们应用我们的工作流程来识别关键的肌苷异构体关联，以帮助阐明 ADAR 在肿瘤发生中的重要性。最终，我们发现长读方法为表征 RNA 变异和剪接模式之间的关系提供了有价值的见解。