Department of Proteomic and Genomic Technologies, Genentech, South San Francisco, CA, USA.
Department of Discovery Oncology, Genentech, South San Francisco, CA, USA.
Nat Commun. 2024 Aug 12;15(1):6916. doi: 10.1038/s41467-024-51252-6.
Single-cell RNA sequencing predominantly employs short-read sequencing to characterize cell types, states and dynamics; however, it is inadequate for comprehensive characterization of RNA isoforms. Long-read sequencing technologies enable single-cell RNA isoform detection but are hampered by lower throughput and unintended sequencing of artifacts. Here we develop Single-cell Targeted Isoform Long-Read Sequencing (scTaILoR-seq), a hybridization capture method which targets over a thousand genes of interest, improving the median number of on-target transcripts per cell by 29-fold. We use scTaILoR-seq to identify and quantify RNA isoforms from ovarian cancer cell lines and primary tumors, yielding 10,796 single-cell transcriptomes. Using long-read variant calling we reveal associations of expressed single nucleotide variants (SNVs) with alternative transcript structures. Phasing of SNVs across transcripts enables the measurement of allelic imbalance within distinct cell populations. Overall, scTaILoR-seq is a long-read targeted RNA sequencing method and analytical framework for exploring transcriptional variation at single-cell resolution.
单细胞 RNA 测序主要采用短读测序来描述细胞类型、状态和动态；然而，它不足以全面描述 RNA 异构体。长读测序技术能够检测单细胞 RNA 异构体，但受到通量较低和非预期的人工制品测序的阻碍。在这里，我们开发了单细胞靶向异构体长读测序（scTaILoR-seq），这是一种杂交捕获方法，靶向一千多个感兴趣的基因，将每个细胞的目标转录本中位数提高了 29 倍。我们使用 scTaILoR-seq 从卵巢癌细胞系和原发性肿瘤中识别和定量 RNA 异构体，产生了 10796 个单细胞转录组。使用长读变异 calling，我们揭示了表达的单核苷酸变异（SNV）与替代转录结构的关联。跨越转录本的 SNV 相位能够在不同的细胞群体中测量等位基因失衡。总的来说，scTaILoR-seq 是一种长读靶向 RNA 测序方法和分析框架，用于在单细胞分辨率下探索转录变异。
