Hybrid untargeted and targeted RNA sequencing facilitates genotype-phenotype associations at single-cell resolution

Long-read scRNA-seq has enabled simultaneous, unbiased identification of transcriptomic variants and expression profiling. However, it often suffers from limited read coverage, which constrains its ability to link genotype and phenotype at single-cell resolution. To address this, we evaluated multiple single-cell sequencing technologies for cell typing and genotyping: short-read whole-transcriptome amplification (SR-WTA) via Illumina, long-read whole-transcriptome amplification (LR-WTA), and long-read targeted sequencing (LR-Twist) via the Pacific Biosciences platform. Based on these evaluations, we propose a hybrid strategy with an accompanying Snakemake pipeline that integrates SR-WTA with LR-Twist to leverage the strengths of both approaches. SR-WTA provides broad transcriptomic coverage and allows detection of low-RNA-content cell types, while LR-Twist enriches a targeted panel of 50 genes to achieve deeper coverage for calling variants. By linking single-cell genotypic and transcriptomic states, this hybrid strategy enhances the resolution and statistical power to investigate how mutations shape cellular transcriptional programs.