Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics

Abstract

Despite rapid developments in single cell sequencing, sample-specific batch effects, detection of cell multiplets, and experimental costs remain outstanding challenges. Here, we introduce Cell Hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its original sample, robustly identify cross-sample multiplets, and “super-load” commercial droplet-based systems for significant cost reduction. We validate our approach using a complementary genetic approach and demonstrate how hashing can generalize the benefits of single cell multiplexing to diverse samples and experimental designs.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1603-1) contains supplementary material, which is available to authorized users.

Vireo: Bayesian demultiplexing of pooled single-cell RNA-seq data without genotype reference

“…The Vireo software provides helper functions for designing such experiments, which directly leverages the reconstructed genotypes in the pool to define a small set of discrimatory variants (Methods). Molecular barcoding strategies, e.g., [9,10,11,12], have recently emerged as an alternatives to genetic barcoding in many respects courtesy of their more universal applicability. For example, molecular barcoding enables pooling multiple treatment conditions or tissues from the same individual or from individuals with the same genetic background (e.g.…”

“…In addition to simpli-fied experimental logistics and reduced batch variation, pooled designs can also facilitate the identification of double cells. Existing barcoding strategies include molecular labelling prior to analysis [9,10,11,12] as well as exploiting natural genetic barcodes of germline variants that segregate between pooled individuals [13]. While molecular barcoding is in principle applicable to any study design, genetic barcoding is both elegant and can be seamlessly integrated in existing scRNA-seq workflows, without the need to introduce additional processing steps.…”

Inference and effects of barcode multiplets in droplet-based single-cell assays

“…While our existing computational approach (bap) can facilitate the identification of barcode multiplets in scATAC-seq data, further experimental and computational tools are needed to more broadly identify these effects in RNA or genome sequencing droplet-based assays. We envision a combination of dense exogenous barcodes via cell hashing 10 and evolved by CRISPR-Cas9 11 or intrinsic features such as clonal mutations, rearrangements, or highly correlated abundances with barcode sequence similarity metrics could be leveraged to better infer barcode multiplets. Such approaches would complement existing tools that robustly identify cell doublets 12,13 and empty droplets 14 from droplet-based scRNA-seq and further mitigate hidden confounders in single-cell data.…”

Solo: doublet identification via semi-supervised deep learning

“…These protocols isolate single cells by encapsulating them in droplets that contain identifying barcodes. Droplets collect cells according to the Poisson distribution; thus, they encapsulate multiple cells at a rate dependent on the concentration of loaded cells [10,11]. Instances of multiple cells to a single barcode, referred to as doublets (or multiplets), can depict nonexistent transcriptional profiles that impair and mislead biological inferences from downstream analyses such as low dimensional visualization, clustering, and differential expression ( Fig.…”