Defining Cell Identity with Single-Cell Omics

Abstract

Cells are a fundamental unit of life, and the ability to study the phenotypes and behaviors of individual cells is crucial to understanding the workings of complex biological systems. Cell phenotypes (epigenomic, transcriptomic, proteomic, and metabolomic) exhibit dramatic heterogeneity between and within the different cell types and states underlying cellular functional diversity. Cell genotypes can also display heterogeneity throughout an organism, in the form of somatic genetic variation-most notably in the emergence and evolution of tumors. Recent technical advances in single-cell isolation and the development of omics approaches sensitive enough to reveal these aspects of cell identity have enabled a revolution in the study of multicellular systems. In this review, we discuss the technologies available to resolve the genomes, epigenomes, transcriptomes, proteomes, and metabolomes of single cells from a wide variety of living systems.

Keywords: epigenomics; genomics; proteomics; single-cell; technology; transcriptomics.

Figure 1

Definitions of cell type and state. A) In the hematopoietic system, cell types have been typically defined by a combination of cell surface marker expression and functional output in in vitro and in vivo assays. B) Within cell types, multiple cell states are possible, including quiescence, active cycling, senescence, and in some cases, resting and activated states. C) Population‐level characterization enables molecular definition of the differences between cell types, in this hypothetical example using principal components analysis (PCA) of RNA‐seq data. This, however, does not reveal heterogeneity within these phenotypically defined populations. Through (D) single‐cell analysis, it is possible to explore this heterogeneity, even in rare cell populations such as HSCs, revealing novel cell phenotypes—cell types and states—within a “homogeneous” population of cells. Abbreviations: LT‐HSC, long‐term reconstituting HSC; FSR‐HSC, finite self‐renewal HSC; LMPP, lymphoid‐primed multipotential progenitors; CMP, common myeloid progenitor; MK, megakaryocyte; E, eryrthroid; My, Myeloid; T, T‐cell; B, B‐cell; PC, principal component.

Figure 2

Methods for isolation and sequencing of single cells. A) FACS‐based cell isolation enables selective deposition of single cells into multiwell plates for downstream molecular processing. Index sorting allows some information about each cell's phenotype to be recorded as it is deposited into the well. Once the cells have been deposited, a number of molecular processes are possible. B) Droplet‐based cell isolation involves the partitioning of single cells into individual droplets with uniquely barcoded oligonucleotides. In the case of single‐cell mRNA‐seq these barcoded oligos prime first strand synthesis of cDNA from the poly‐A tail. Reverse transcription is then performed in a droplet emulsion, resulting in each cDNA molecule being uniquely tagged based on its cell of origin. Unique molecular identifiers (UMIs) are also incorporated to enable unequivocal counting of the number of detected molecules. C) Nanowell‐based approaches use a similar approach, but rather than partitioning cells into droplets, cells are captured in minute wells with uniquely barcoded beads. D) Combinatorial indexing strategies have used a two‐step barcoding strategy for DNA or cDNA molecules to increase throughput without the need for microfluidics. First, a primary barcode is added to small pools of FACS isolated cells/nuclei (in the case of cDNA, this is added during reverse transcription, in the case of DNA this is added through tagmentation with barcoded adaptors) which are then re‐pooled with other distinctly barcoded cells and again sorted into small pools, where they received a second barcode. Thus, each cell receives a unique pairing of barcoded molecules, enabling each sequencing read to be assigned to an individual cell.

Figure 3

Methods for the analysis of single‐cell identity. An overview of the methods currently available to study the genome, epigenome, transcriptome and proteomes of single cells, some of which have been combined into multi‐omic single‐cell assays.