Haplotype-resolved full-length transcriptome analysis in single cells by scCirRL-seq
We report scCirRL-seq (single-cell Circularized Re-Linearized sequencing), a novel nanopore-based long-read single-cell RNA-seq (scRNA-seq) strategy and its companion computational workflow for haplotype-resolved full-length transcriptome analysis in single cells. By incorporating a circularization and re-linearization step of full-length cDNAs during library preparation, scCirRL-seq mitigates common artifacts in long-read scRNA-seq libraries, producing over 70% more usable reads than standard nanopore long-read scRNA-seq. We generated 853.6 million scCirRL-seq long reads from 51,331 peripheral blood mononuclear cells as well as matching whole-genome sequencing (WGS) data of 13 adult donors. We identified cell-type-specific isoform variations along with their potential regulators in major immune cell types. By integrating scCirRL-seq and WGS data, we discovered 545 unique genes exhibiting allele-specific variations in transcript isoform usage in specific immune cell types. These events were significantly enriched for genetic variants linked to genome-wide association study signals of human traits and diseases (210 out of 545). We highlighted multiple examples, including allele-specific isoform variation of OAS1 in monocytes, that was linked to SARS-CoV-2 infection and COVID-19 severity. Collectively, we demonstrate that scCirRL-seq provides an effective and broadly applicable strategy for elucidating alternative isoform variations and their genetic regulation in single cells.