The high diversity and complexity of the eukaryotic transcriptome make it difficult to effectively detect specific transcripts of interest. Current targeted RNA sequencing methods often require complex pre-sequencing enrichment steps, which can compromise the comprehensive characterization of the entire transcriptome. Here we describe programmable full-length isoform transcriptome sequencing (PROFIT-seq), a method that enriches target transcripts while maintaining unbiased quantification of the whole transcriptome. PROFIT-seq employs combinatorial reverse transcription to capture polyadenylated, non-polyadenylated and circular RNAs, coupled with a programmable control system that selectively enriches target transcripts during sequencing. This approach achieves over 3-fold increase in effective data yield and reduces the time required for detecting specific pathogens or key mutations by 75%. We applied PROFIT-seq to study colorectal polyp development, revealing the intricate relationship between host immune responses and bacterial infection. PROFIT-seq offers a powerful tool for accurate and efficient sequencing of target transcripts while preserving overall transcriptome quantification, with broad applications in clinical diagnostics and targeted enrichment scenarios.

真核生物转录组的高度多样性和复杂性使得难以有效检测感兴趣的特异性转录本。目前的靶向 RNA 测序方法通常需要复杂的测序前富集步骤，这可能会影响整个转录组的全面表征。在这里，我们描述了可编程的全长亚型转录组测序 （PROFIT-seq），这是一种在保持整个转录组的无偏倚定量的同时富集靶转录本的方法。PROFIT-seq 采用组合逆转录来捕获多聚腺苷酸化、非多聚腺苷酸化和环状 RNA，并结合可编程控制系统，在测序过程中选择性地富集靶标转录本。这种方法将有效数据产量提高了 3 倍以上，并将检测特定病原体或关键突变所需的时间缩短了 75%。我们应用 PROFIT-seq 研究结直肠息肉的发育，揭示了宿主免疫反应与细菌感染之间的复杂关系。PROFIT-seq 为准确、高效地对靶转录本进行测序提供了强大的工具，同时保留了整体转录组定量，在临床诊断和靶向富集场景中具有广泛的应用。