Reference-free multiplexed single-cell sequencing identifies genetic modifiers of the human immune response

Multiplexed single-cell sequencing (mux-seq) using single-nucleotide polymorphisms (SNPs) has emerged as an efficient approach to perform expression quantitative trait loci (eQTL) studies that map interactions between genetic variants and cell types, cell states, or experimental perturbations. Here we introduce the clue framework, a novel approach to encode mux-seq experiments that eliminates the need for reference genotypes and experimental barcoding. The clue framework is made possible by the development of freemuxlet, an algorithm that clusters cells based on SNPs called from single-cell RNA-seq or ATAC-seq data. To demonstrate the feasibility of clue, we profiled the surface protein and RNA abundances of peripheral blood mononuclear cells from 64 individuals, stimulated with 5 distinct extracellular stimuli -- all within a single day. Our analysis of the demultiplexed data identified rare immune cell types and cell type-specific responses to interferon and toll-like receptor stimulation. Furthermore, by integrating genotyping data, we mapped response eQTLs specific to certain cell types. These findings showcase the potential and scalability of the clue framework for reference-free multiplexed single-cell sequencing studies.