Acquired treatment resistance in homologous recombination (HR)-deficient high-grade serous ovarian carcinoma (HGSOC) commonly involves restoration of HR, often through secondary somatic reversion mutations in HR genes such as BRCA1, BRCA2 and BRIP1. Recent work suggests that these resistance mechanisms are frequently subclonal, raising the question of how reversion-negative cells persist during treatment. To address this, we developed a single-cell sequencing approach that enables direct genotyping of individual cells and links genotype to transcriptomic phenotype. In a pilot experiment, we analysed two HGSOC cell lines with known reversion mutations by combining short-read whole-transcriptome single-cell RNA sequencing with long-read targeted single-cell cDNA sequencing on an Oxford Nanopore MinION using a 44-gene hybridisation capture panel. A custom bioinformatics pipeline incorporating Flexiplex resolved multiallelic events and annotated cell barcodes with driver, reversion and TP53 mutations. Genotypes were then transferred to the short-read dataset following outlier and doublet filtering, enabling differential expression analysis between cells with and without reversions. We have also applied this protocol to patient ascites and tumour samples collected before and after treatment. We successfully detected driver and reversion alleles at single-cell resolution in both cell lines. Rare wild-type heterozygous cells were also identified, suggesting back-reversion events or subclones that escaped loss of heterozygosity and would likely be missed by bulk sequencing. This approach enables detection of subclonal resistance mechanisms and characterisation of transcriptional programs associated with persistence of non-reverted cells in HR-deficient HGSOC.

Co-authors: Stuart Bencraig, David Yoannidis, Madelynne Willis, Joy Hendley, Timothy Semple, Alicia Oshlack, Elizabeth Christie