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

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Single-cell long-read sequencing offers insights into isoform usage and cellular mutation heterogeneity.
• Low sequencing throughput in current methods limits mutation calling for specific genes.

Purpose of the Study:

• To develop a highly specific and efficient method for targeted transcript capture in single-cell long-read sequencing.
• To enable in-depth analysis of mutation status and transcript usage for genes of interest.

Main Methods:

• Developed single-cell Rapid Capture Hybridization sequencing (scRaCH-seq) using a probe panel for transcript capture.
• Utilized barcoded primers for pooling and Oxford Nanopore Technologies sequencing.
• Applied scRaCH-seq to stored single-cell cDNA, enabling integration with existing short-read RNA-seq data.

Main Results:

• scRaCH-seq demonstrated high specificity and efficiency in capturing targeted transcripts.
• Successfully detected SF3B1 isoforms and mutations with high sensitivity in chronic lymphocytic leukemia (CLL) samples.
• Integrated scRaCH-seq with scRNA-seq data revealed gene expression differences in SF3B1-mutated CLL cells.

Conclusions:

• scRaCH-seq is a powerful tool for analyzing long-read transcripts of multiple genes in single-cell genomics.
• The method facilitates sensitive mutation detection and isoform analysis, overcoming limitations of low read coverage.
• scRaCH-seq enables combined analysis with existing scRNA-seq data for comprehensive genomic insights.