Scalable genotyping in fixed transcriptomes resolves clonal heterogeneity via single-cell sequencing

Single-cell transcriptomics has revolutionized our understanding of heterogeneous cell populations. However, technical limitations of widely-used platforms have limited our ability to link transcriptional states to somatic mutations within the same cells at scale. Here, we introduce Genotyping in Fixed Transcriptomes (GIFT), a novel assay for simultaneous detection of hundreds of targeted genetic variants and whole transcriptome profiles in single cells. The core innovation of GIFT is a rationally designed gapfilling reaction between adjacent single-stranded DNA (ssDNA) probes that barcodes native transcript sequence to enable highly-specific targeted mutation detection. GIFT achieves >99% genotyping accuracy and flexible capture of hundreds of mutations per cell, including in FFPE (Formalin-Fixed Paraffin-Embedded) tissue, enabling clonal lineage tracing in heterogeneous settings. We demonstrate the unique scalability of GIFT by profiling >700,000 cells from 35 donors with myeloproliferative neoplasms (MPN), revealing mutation-dependent hematopoietic responses to systemic inflammation associated with the characteristic JAK2V617 mutation, including an allelic dose gradient of interferon-associated transcriptional programs and transcriptional priming of hematopoietic stem cells that develop into divergent disease states. Together, the unique technical advantages of GIFT enable direct resolution of genotype-to-phenotype relationships via clonal lineage tracing with comprehensive cell state measurements at single-cell resolution.