Single-cell Rapid Capture Hybridization sequencing reliably detects isoform usage and coding mutations in targeted genes

Single-cell long-read sequencing has transformed our understanding of isoform usage and the mutation heterogeneity between cells. Despite unbiased in-depth analysis, the low sequencing throughput often results in insufficient read coverage, thereby limiting our ability to perform mutation calling for specific genes. Here, we developed a single-cell Rapid Capture Hybridization sequencing (scRaCH-seq) method that demonstrates high specificity and efficiency in capturing targeted transcripts using long-read sequencing, allowing an in-depth analysis of mutation status and transcript usage for genes of interest. The method includes creating a probe panel for transcript capture, using barcoded primers for pooling and efficient sequencing via Oxford Nanopore Technologies platforms. scRaCH-seq is applicable to stored and indexed single-cell cDNA, which allows analysis to be combined with existing short-read RNA-seq data sets. In our investigation of <i>BTK</i> and <i>SF3B1</i> genes in samples from patients with chronic lymphocytic leukemia (CLL), we detect <i>SF3B1</i> isoforms and mutations with high sensitivity. Integration with short-read single-cell RNA sequencing (scRNA-seq) data reveals significant gene expression differences in <i>SF3B1</i>-mutated CLL cells, although it does not impact the sensitivity of the anticancer drug venetoclax. scRaCH-seq's capability to study long-read transcripts of multiple genes makes it a powerful tool for single-cell genomics.