Long-read RNA sequencing (RNA-seq) is a powerful technology for transcriptome analysis, but the relatively low throughput of current long-read sequencing platforms limits transcript coverage. One strategy for overcoming this bottleneck is targeted long-read RNA-seq for preselected gene panels. We present TEQUILA-seq, a versatile, easy-to-implement, and low-cost method for targeted long-read RNA-seq utilizing isothermally linear-amplified capture probes. When performed on the Oxford nanopore platform with multiple gene panels of varying sizes, TEQUILA-seq consistently and substantially enriches transcript coverage while preserving transcript quantification. We profile full-length transcript isoforms of 468 actionable cancer genes across 40 representative breast cancer cell lines. We identify transcript isoforms enriched in specific subtypes and discover novel transcript isoforms in extensively studied cancer genes such as TP53. Among cancer genes, tumor suppressor genes (TSGs) are significantly enriched for aberrant transcript isoforms targeted for degradation via mRNA nonsense-mediated decay, revealing a common RNA-associated mechanism for TSG inactivation. TEQUILA-seq reduces the per-reaction cost of targeted capture by 2-3 orders of magnitude, as compared to a standard commercial solution. TEQUILA-seq can be broadly used for targeted sequencing of full-length transcripts in diverse biomedical research settings.

长读长 RNA 测序 (RNA-seq) 是一种强大的转录组分析技术，但当前长读长测序平台的通量相对较低，限制了转录本覆盖范围。克服这一瓶颈的一种策略是针对预选基因组进行长读长 RNA 测序。我们推出了 TEQUILA-seq，这是一种利用等温线性扩增捕获探针进行靶向长读长 RNA 测序的多功能、易于实施且低成本的方法。当在具有不同大小的多个基因组的牛津纳米孔平台上执行时，TEQUILA-seq 一致且显着地丰富了转录本覆盖范围，同时保留了转录本定量。我们分析了 40 个代表性乳腺癌细胞系中 468 个可作用癌症基因的全长转录亚型。我们鉴定了富含特定亚型的转录亚型，并在广泛研究的癌症基因（例如TP53）中发现了新的转录亚型。在癌症基因中，肿瘤抑制基因 (TSG) 显着富集异常转录亚型，这些转录亚型的目标是通过 mRNA 无义介导的衰变进行降解，揭示了 TSG 失活的常见 RNA 相关机制。与标准商业解决方案相比，TEQUILA-seq 将靶向捕获的每次反应成本降低了 2-3 个数量级。TEQUILA-seq 可广泛用于多种生物医学研究环境中的全长转录本的靶向测序。