Sample-multiplexing approaches for single-cell sequencing

Abstract

Single-cell sequencing is widely used in biological and medical studies. However, its application with multiple samples is hindered by inefficient sample processing, high experimental costs, ambiguous identification of true single cells, and technical batch effects. Here, we introduce sample-multiplexing approaches for single-cell sequencing in transcriptomics, epigenomics, genomics, and multiomics. In single-cell transcriptomics, sample multiplexing uses variants of native or artificial features as sample markers, enabling sample pooling and decoding. Such features include: (1) natural genetic variation, (2) nucleotide-barcode anchoring on cellular or nuclear membranes, (3) nucleotide-barcode internalization to the cytoplasm or nucleus, (4) vector-based barcode expression in cells, and (5) nucleotide-barcode incorporation during library construction. Other single-cell omics methods are based on similar concepts, particularly single-cell combinatorial indexing. These methods overcome current challenges, while enabling super-loading of single cells. Finally, selection guidelines are presented that can accelerate technological application.

Keywords: Cell Hashing; Multi-omics; Spatial transcriptomics; scATAC-seq; scRNA-seq.

Fig. 1

A Hierarchy of sample-multiplexing approaches used for single-cell sequencing. The different shapes represent different strategies, which are also mentioned in (B). B Timeline of sample-multiplexing approaches for single-cell sequencing. Omics targeted are distinguished by various colors. The strategies are represented by different shapes

Fig. 2

Schematic overview of five sample-multiplexing strategies used for scRNA-seq. A Natural genetic variation. Without additional labeling, computational demultiplexing is conducted based on SNPs. B Nucleotide-barcode anchoring on cellular or nuclear membranes. The example shown here is Cell Hashing, where oligo-tagged antibodies (hashtags) bind to ubiquitously expressed cell-surface proteins. Oligos with a poly (A) tail are captured along with mRNA. Cells can be assigned to their sample of origin based on different barcodes in the hashtags. C Nucleotide-barcode internalization into the cytoplasm or nucleus. Barcoded DNA traverses the cellular or nuclear membrane by liposomal transfection or directly diffuses into the nuclei. SBO: short barcode oligonucleotide. D Vector-based barcode expression in cells. E Nucleotide-barcode incorporation during library construction

Fig. 3

Main processes during library construction of the “sci family.” Each round of indexing provides an opportunity for sample multiplexing to occur, especially in round 1. With the inherent ability of sample multiplexing, combinatorial indexing occupies an important position in multiplexed single-cell sequencing

Fig. 3

Main processes during library construction of the “sci family.” Each round of indexing provides an opportunity for sample multiplexing to occur, especially in round 1. With the inherent ability of sample multiplexing, combinatorial indexing occupies an important position in multiplexed single-cell sequencing

Fig. 4

Representative applications of sample-multiplexing approaches for single-cell sequencing