Scalable genotyping in fixed transcriptomes resolves clonal heterogeneity via single-cell sequencing
Key claim: A new single-cell method (GIFT) simultaneously captures somatic mutations and whole transcriptomes at scale, revealing that JAK2V617F allele dosage drives a gradient of interferon-associated transcriptional programs and hematopoietic stem cell priming toward divergent MPN disease states.
Abstract
Despite the promise of single-cell transcriptomics for understanding cell states in heterogeneous populations, widely used platforms have limited ability to link transcriptional states to somatic mutations within the same cells. Here, we introduce Genotyping in Fixed Transcriptomes (GIFT) for the simultaneous detection of large numbers of targeted genetic variants with 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 greater than 99% genotyping accuracy and flexible capture of hundreds of mutations per cell, including in formalin-fixed, paraffin-embedded (FFPE) tissue, enabling clonal lineage tracing in heterogeneous settings. We demonstrate the unique scalability of GIFT by profiling more than 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 priming of hematopoietic stem cells that develop into divergent disease states. The technical advantages of GIFT enable direct resolution of genotype-to-phenotype relationships via clonal tracing with comprehensive cell-state measurements at single-cell resolution.
Why this matters
Subtype assignment in JAK2V617F-driven MPNs has long been correlated with allele burden (ET < PV < PMF), but the cellular mechanism has been unresolved. GIFT’s simultaneous single-cell genotyping and whole-transcriptome readout reframes this as a quantitative, dose-dependent interferon-program gradient in hematopoietic stem cells that primes divergent disease trajectories — providing a unifying biological axis across ET, PV, and PMF and a potential biomarker substrate for predicting transformation and IFNα responsiveness.