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

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Single-cell transcriptomics offers insights into cell heterogeneity.
• Linking transcriptional states to somatic mutations in single cells remains a challenge.
• Existing platforms have limitations in simultaneous genetic and transcriptomic profiling.

Purpose of the Study:

• Introduce Genotyping in Fixed Transcriptomes (GIFT), a novel assay.
• Enable simultaneous detection of genetic variants and transcriptome profiles in single cells.
• Facilitate clonal lineage tracing and genotype-to-phenotype relationship resolution.

Main Methods:

• Developed a novel assay (GIFT) using gapfilling reactions between ssDNA probes.
• Barcodes native transcript sequences for highly specific mutation detection.
• Achieved high genotyping accuracy (>99%) and flexible capture of hundreds of mutations per cell.
• Applied GIFT to profile over 700,000 cells from myeloproliferative neoplasm (MPN) patients.

Main Results:

• Demonstrated GIFT's scalability and accuracy, including in FFPE tissues.
• Revealed mutation-dependent hematopoietic responses to inflammation in MPNs.
• Identified an allelic dose gradient of interferon-associated programs linked to JAK2V617.
• Showcased GIFT's ability to trace clonal lineages and link genotype to phenotype.

Conclusions:

• GIFT overcomes technical limitations in single-cell mutation and transcriptome analysis.
• Enables comprehensive cell state measurements and clonal lineage tracing at single-cell resolution.
• Provides new insights into genotype-to-phenotype relationships in complex diseases like MPNs.