Probing infectious disease by single-cell RNA sequencing: Progresses and perspectives

Abstract

The increasing application of single-cell RNA sequencing (scRNA-seq) technology in life science and biomedical research has significantly increased our understanding of the cellular heterogeneities in immunology, oncology and developmental biology. This review will summarize the development of various scRNA-seq technologies; primarily discussing the application of scRNA-seq on infectious diseases, and exploring the current development, challenges, and potential applications of scRNA-seq technology in the future.

Keywords: 3C, Chromosome Conformation Capture; ACE2, Angiotensin-Converting Enzyme 2; ARDS, acute respiratory distress syndrome; ATAC-seq, Assay for Transposase-Accessible Chromatin using sequencing; BCR, B cell receptor; CEL-seq, Cell Expression by Linear amplification and Sequencing; CLU, clusterin; COVID-19, corona virus disease 2019; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; CytoSeq, gene expression cytometry; DENV, dengue virus; FACS, fluorescence-activated cell sorting; GNLY, granulysin; GO analysis, Gene Ontology analysis; HIV, Human Immunodeficiency Virus; IAV, Influenza A virus; IGHV/HD/HJ/HC, Immune globulin heavy V/D/J/C/ region; IGLV/LJ/LC, Immune globulin light V/J/C/ region; ILC, Innate Lymphoid Cell; Infectious diseases; LIGER, Linked Inference of Genomics Experimental Relationships; MAGIC, Markov Affinity-based Graph Imputation of Cells; MARS-seq, Massively parallel single-cell RNA sequencing; MATCHER, Manifold Alignment To CHaracterize Experimental Relationships; MCMV, mouse cytomegalovirus; MERFISH, Multiplexed, Error Robust Fluorescent In Situ Hybridization; MLV, Moloney Murine Leukemia Virus; MOFA, Multi-Omics Factor Analysis; MOI, multiplicity of infection; PBMCs, peripheral blood mononuclear cells; PLAC8, placenta-associated 8; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SAVER, Single-cell Analysis Via Expression Recovery; SPLit-seq, split pool ligation-based tranome sequencing; STARTRAC, Single T-cell Analysis by RNA sequencing and TCR TRACking; STRT-seq, Single-cell Tagged Reverse Transcription sequencing; Single-cell RNA sequencing; TCR, T cell receptor; TSLP, thymic stromal lymphopoietin; UMAP, Uniform Manifold Approximation and Projection; UMI, Unique Molecular Identifier; mcSCRB-seq, molecular crowding single-cell RNA barcoding and sequencing; pDCs, plasmacytoid dendritic cells; scRNA-seq, single cell RNA sequencing technology; sci-RNA-seq, single-cell combinatorial indexing RNA sequencing; seqFISH, sequential Fluorescent In Situ Hybridization; smart-seq, switching mechanism at 5′ end of the RNA transcript sequencing; t-SNE, t-Distributed stochastic neighbor embedding.

Fig. 1

The blue boxes represent significant events in the history of scRNA-seq development. The remaining color boxes are abbreviations of various technologies. The black box represents microfluidics-based technology. The red boxes represent plate-based technology. Green boxes represent microdroplet-based technology. Yellow box represents nanowell-arrays-based technology. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Immune atlas study. (A) Identifying novel immune cell subtypes; (B) Detecting immune cell landscape during infection; (C) Detecting changes of inflammatory responses; (D) Identifying immune signaling pathways for differentially expressed genes during infection.

Fig. 3

Study the host-pathogen interaction. (A) Identifying susceptible cell types; (B) Studying infection dynamics. Each curve represents the expression of viral genes contained in cells at varying levels of MOI.

Fig. 4

TCR and BCR sequencing. (A) Antibody Structure; (B) BCR gene structure, including IGLV, IGLJ, IGLC, IGHV, IGHD, IGHJ, IGHC (IG-, Immune Globulin-, immunoglobulin, LV / LJ / LC / HV / HD / HJ / HC indicates the light chain variable region / light chain binding region / light chain constant region / heavy chain variable region / heavy chain multivariable region / heavy chain binding region / heavy chain constant region respectively). Colors reflect structural regions shown in A and B; (C) Structure of the TCR; (D) TCR gene structure. Colors reflect structural regions shown in A and B; (E) Standard procedure for BCR / TCR sequencing; (F) Changes in TCR composition before and after infection.

Fig. 5

Several emerging single-cell sequencing methods. (A) Including ATAC-seq, whole transcriptome analysis, immune repertoire study, CRISPR-I and methylation sequencing; (B) Schematic diagram of a spatial transcriptome sequencing chips. There are four chips for capturing single-cell mRNA on a slice. Each spot in the chip contains an oligonucleotide sequence, which includes poly (dT), UMI, spatial barcode and partial read 1. Partial read 1 includes 22nt for Illumina sequencing, spatial barcode includes 16nt 10X barcode, and UMI includes 12nt unique molecular identifier.