Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.

中文翻译：

---

FLAME：用于综合剪接组表征的长读生物信息学工具

使用纳米孔测序对差异剪接的 RNA 转录本进行综合表征受到依赖于现有注释的生物信息学工具的限制。我们开发了 FLAME，这是一种生物信息学管道，用于对基因特异性或转录组范围的长读长测序数据进行选择性剪接分析。FLAME 是一种基于 Python 的工具，旨在提供对全长剪接变体的可理解量化、对剪接位点和外显子的可靠从头识别以及以加权邻接矩阵形式表示的连续外显子连接。值得注意的是，该工作流程避免了与参考注释不足相关的问题，并允许纳入短读长测序数据以提高纳米孔测序读长的置信度。在这项研究中，Epstein-Barr 病毒的长链非编码 RNARPMS1用于演示管道的效用。RPMS1在 Epstein-Barr 病毒相关癌症中普遍表达，并且已知会经历大量差异剪接。为了完全解析RPMS1剪接组，我们将来自原发性胃腺癌和鼻咽癌细胞系的基因特异性纳米孔测序读数与匹配的公开可用短读长测序数据集相结合。使用 FLAME 检测到所有先前报告的剪接变体，包括推定的 ORF。此外，在RPMS1基因中发现了 32 个新的外显子，包括两个内含子保留和一个盒式外显子。