Single-cell Rapid Capture Hybridization sequencing to reliably detect isoform usage and coding mutations in targeted genes
- Hongke Peng1,
- Jafar S. Jabbari1,
- Luyi Tian1,
- Changqing Wang1,
- Yupei You1,
- Chong Chyn Chua2,
- Natasha S. Anstee1,
- Noorul Amin1,
- Andrew H. Wei3,
- Nadia Davidson1,
- Andrew W. Roberts3,
- David Huang1,
- Matthew E Ritchie1 and
- Rachel Thijssen4,5
- 1 The Walter and Eliza Hall Institute of Medical Research, University of Melbourne;
- 2 The Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Monash Health, Northern Health;
- 3 The Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Royal Melbourne Hospital and Peter MacCallum Cancer Centre;
- 4 The Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Amsterdam UMC, Cancer Center Amsterdam
Abstract
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 demonstrated 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 datasets. In our investigation of BTK and SF3B1 genes in samples from patients with chronic lymphocytic leukaemia (CLL), we detected SF3B1 isoforms and mutations with high sensitivity. Integration with short-read scRNA-seq data revealed significant gene expression differences in SF3B1-mutated CLL cells, though it did not impact the sensitivity of the anti-cancer drug venetoclax. scRaCH-seq's capability to study long-read transcripts of multiple genes makes it a powerful tool for single-cell genomics.
- Received March 13, 2024.
- Accepted December 10, 2024.
- Published by Cold Spring Harbor Laboratory Press
This manuscript is Open Access.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International license), as described at http://creativecommons.org/licenses/by-nc/4.0/.
