Wu et al.

Biomarker Research (2024) 12:110 Biomarker Research

https://doi.org/10.1186/s40364-024-00643-4

REVIEW Open Access

Single-cell sequencing to multi-omics:

technologies and applications
Xiangyu Wu1†, Xin Yang1†, Yunhan Dai2, Zihan Zhao1, Junmeng Zhu3, Hongqian Guo1* and Rong Yang1*

Abstract
Cells, as the fundamental units of life, contain multidimensional spatiotemporal information. Single-cell RNA
sequencing (scRNA-seq) is revolutionizing biomedical science by analyzing cellular state and intercellular
heterogeneity. Undoubtedly, single-cell transcriptomics has emerged as one of the most vibrant research fields
today. With the optimization and innovation of single-cell sequencing technologies, the intricate multidimensional
details concealed within cells are gradually unveiled. The combination of scRNA-seq and other multi-omics is at the
forefront of the single-cell field. This involves simultaneously measuring various omics data within individual cells,
expanding our understanding across a broader spectrum of dimensions. Single-cell multi-omics precisely captures
the multidimensional aspects of single-cell transcriptomes, immune repertoire, spatial information, temporal
information, epitopes, and other omics in diverse spatiotemporal contexts. In addition to depicting the cell atlas
of normal or diseased tissues, it also provides a cornerstone for studying cell differentiation and development
patterns, disease heterogeneity, drug resistance mechanisms, and treatment strategies. Herein, we review traditional
single-cell sequencing technologies and outline the latest advancements in single-cell multi-omics. We summarize
the current status and challenges of applying single-cell multi-omics technologies to biological research and
clinical applications. Finally, we discuss the limitations and challenges of single-cell multi-omics and potential
strategies to address them.
Keywords Single-cell multi-omics, scRNA-seq, scTCR-seq, scBCR-seq, Spatial transcriptomics, Proteomics,
Microbiome, Metabolome, Computational biology

Introduction
Transcriptomic analysis has provided fundamental
insights into the study of gene expression in exploring
the functions related to the process of life development,
†
disease progression, and drug action, etc. Over the past
Xiangyu Wu and Xin Yang contributed equally to this work.
decade, bulk RNA sequencing has shed light on biologi-
*Correspondence: cal functions from a pooled cell population transcrip-
Hongqian Guo
[email protected] tomic perspective. However, it represents an average
Rong Yang across the myriad of cells within a tissue, merely reflect-
[email protected] ing the characteristics of cell populations or perhaps pre-
1
Department of Urology, Nanjing Drum Tower Hospital, Affiliated Hospital
of Medical School, Nanjing University, 321 Zhongshan Road, dominantly the information of the most numerous cells.
Nanjing 210008, Jiangsu, China Moreover, bulk RNA sequencing neither elucidates the
2
Medical School, Nanjing University, Nanjing, China variations of a sample at the single-cell level nor reflects
3
Department of Oncology, Nanjing Drum Tower Hospital, Affiliated
Hospital of Medical School, Nanjing University, Nanjing, China the expression levels of rare cells.

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Wu et al. Biomarker Research (2024) 12:110 Page 2 of 28

To tackle these constraints, scRNA-seq has revolution- single-cell analysis, providing technical support to unveil
ized biomedical science by single-cell expression pro- the secrets of cells.
filing. This technology enables detailed exploration of Single-cell multi-omics technologies have emerged.
genetic information at the cellular level across various They refer to the simultaneous measurement of various
tissues and diseases, capturing the inherent heterogeneity types of data in the same cell, allowing for an accurate
within samples. Since Tang et al. pioneered sequencing and detailed depiction of the cellular state. Integrating
technology on a single cell in 2009 [1], the methodology single-cell transcriptomic sequencing with comprehen-
has undergone continuous refinement and maturation. sive multi-omics data to map their inherent connections
These advancements have facilitated the development represents a critical and inevitable trend toward a more
of full-length transcriptome profiling, high-throughput nuanced, multidimensional understanding of life devel-
capabilities, and high-sensitivity scRNA-seq [2–4]. opment and the mechanisms underlying diseases.
Although well-established scRNA-seq has achieved These cutting-edge methods break through the limita-
great success and wide applications in the research field, tions of the conventional scRNA-seq, offering an excit-
it has also triggered new thinking because of its limita- ing solution to explore how cellular modalities affect cell
tions. Cellular information extends well beyond RNA state and function. Single-cell T cell receptor sequencing
sequencing, encompassing the genome, epigenome, pro- (scTCR-seq) [5] and single-cell B cell receptor sequenc-
teome, metabolome, etc., along with crucial details about ing (scBCR-seq) [6] effectively delineate the reper-
spatial relationships and dynamic alterations (Fig. 1B). toires of T and B cells, respectively, which can reveal
Therefore, scientists continually explore new methods for the immune system state. Integration with single-cell
proteomics (CITE-seq) enriches the information with

Fig. 1 (A) Conventional scRNA-seq technologies. (B) Overview of the single-cell multi-omics scheme
Wu et al. Biomarker Research (2024) 12:110 Page 3 of 28

proteomics that is both similarities and discrepancies Seurat, SingleCellExperiment, and SingleR, while
with the transcriptome [7]. Coupled with single-cell assay Python-based representative software includes Scanpy,
for transposase-accessible chromatin using sequencing Loom, and AnnData. Data preprocessing involves imple-
(scATAC-seq), researchers gain insights into chromatin menting data quality control, aligning sequences to ref-
accessibility, identifying active regulatory sequences and erence genomes, and generating expression matrices.
potential transcription factors (TFs). Besides, decipher- Subsequent analyses typically utilize formats like Seurat,
ing temporal and spatial information at the single-cell SingleCellExperiment, AnnData, or Loom. The general
level is fundamental for biological research. Although analysis workflow includes (1) filtering data based on
most temporal data is inferred via computational biology doublets, mitochondrial content, erythrocytes, etc., (2)
technology or scRNA-seq atlas created at multiple time selecting features as highly variable genes, and (3) dimen-
points, the experimental method to unveil newly syn- sion reduction, including principal component analysis
thesized RNA is another way [8]. Spatial transcriptomics (PCA), uniform manifold approximation and projection
technologies merge tissue sectioning with single-cell (UMAP), or t-distributed stochastic neighbor embedding
sequencing to compensate for the inability of scRNA-seq (t-SNE). The advanced analyses aim to answer the bio-
to characterize spatial locations. It is worth mentioning logical questions. Clustering and annotation of cell types
that computational biology is necessary for integrating answer what kinds of cell types are there. At the gene
and analyzing single-cell multi-omics data. The informa- level, differentially expressed genes (DEGs) and gene
tion content varies across different modalities. How to enrichment, which includes gene ontology (GO), kyoto
integrate and process these data is of utmost importance. encyclopedia of genes and genomes (KEGG), and gene
The standard workflow to analyze the multimodal data- set variation analysis (GSVA), aim to identify the differ-
sets is necessary for a broadly applicable strategy of sin- ential genes between cell types or specimens, as well as
gle-cell multi-omics. Additionally, computational biology to clarify their associated pathway profiles. Inferring TFs
methods are already widely employed to simulate related by single-cell regulatory network inference and cluster-
different dimension information. ing (SCENIC) provides the essential gene regulatory net-
The holistic view created by single-cell multi-omics work [12]. At the cell level, cell-cell communication offers
technologies is crucial for understanding the complexi- speculation from the perspective of cellular interactions
ties of biology, providing insights into cellular diversity, [13] while cell trajectory analysis incorporates the tem-
disease mechanisms, and potential therapeutic targets. poral information (Fig. 1A) [14]. Furthermore, inferring
In 2019, single-cell multimodal omics was selected as copy number variations (CNVs) with inferCNV analysis
Method of the Year [9]. In this review, we encapsulate the by comparing gene expression levels in cells with those in
advancements and applications of traditional scRNA-seq, a reference genome is particularly crucial in cancer biol-
outline various single-cell multi-omics methodologies, ogy [15].
and explore their biological and clinical implications, High cost and batch effects remain the major obstacles
while also contemplating current limitations and future for large cohort studies on scRNA-seq. To overcome
directions. these constraints, the integration of multiple samples
for large-scale scRNA-seq analysis has become a preva-
Conventional single-cell sequencing lent practice in research. Batch effects, hampering data
scRNA-seq, stepping onto the stage of history, has com- integration, may arise from different experimental con-
pletely reshaped the study approach of the complexity ditions, such as varying chips, sequencing lanes, or tim-
and heterogeneity within individual cells. scRNA-seq ing of cell processing. Integrating data from multiple
technologies mainly involve microfluidic chips, micro- experiments requires the use of employ algorithms such
droplets, and microwell-based approaches, which as Seurat’s canonical correlation analysis (CCA), mutual
have been well-introduced and compared in previ- nearest neighbors (MNN), or Harmony to batch correc-
ous articles [10, 11]. The main experimental steps of tion [16, 17].
scRNA-seq encompass preparing single-cell suspension, Sample multiplexed scRNA-seq is another solution,
isolating individual cells, capturing their mRNA, con- establishing an efficient method for massively paral-
ducting reverse transcription and nucleic acid amplifica- lel species-mixing experiments [18–22]. Currently, the
tion, and building a transcriptome library (Fig. 1A). predominant approach for this technology involves tag-
Analysis of scRNA-seq via bioinformatics is another ging individual samples with DNA oligonucleotides (oli-
cornerstone for visualizing and understanding the under- gos) barcodes before pooling them together, including
lying patterns and insights within the data. Tools for lipid-tagged DNA [21], chemical cross-linking reaction
analyzing scRNA-seq data are written in a variety of pro- [19], and genetic barcodes [20]. This technology is prag-
gramming languages, with R and Python being the most matic, multiplexing via DNA oligos and demultiplexing
prominent. R-based representative software includes conducted via bioinformatics independently of genetic
Wu et al. Biomarker Research (2024) 12:110 Page 4 of 28

background and sample origin, and is compatible with New advances in single-cell sequencing
other omics technologies, ensuring that the unique bio- Although high-throughput single-cell sequencing has
logical characteristics of individual samples are main- revealed the gene expression characteristics within the
tained. Consequently, it inherently avoids batch effects majority of physiological and diseased cells, the com-
or the loss of biological characteristics post-debatching. plexity of cells extends far beyond its scope. There is
Recently, Zhao et al. reported an improved ClickTags still a need to understand and decode the secrets of cells
method to enable the use in live-cell samples empowered from multiple dimensions. With the thriving develop-
by “click chemistry”, and eliminated the requirement for ment of biology, chemistry, bioinformatics, and other
methanol fixation of samples (Fig. 2B). Moreover, this advanced technologies, researchers are continuously
method has been successfully utilized across various developing new technologies and methods for single-cell
murine cells and human samples of bladder cancer that sequencing. Here, we summarize new advances in sin-
have undergone freeze-thaw cycles, demonstrating its gle-cell sequencing, which can be major trends in future
applicability to diverse single-cell specimens [22]. development.

Fig. 2 A brief method and principle of single-cell multi-omics technologies for (A) scTCR/BCR-seq, (B) ClickTag, (C) proteome, (D) microbiome, (E) me-
tabolome, (F) epigenome
Wu et al. Biomarker Research (2024) 12:110 Page 5 of 28

Temporal information effectively combining computational and biological

One major limitation of current scRNA-seq approaches methods, have gained wide acceptance and popularity,
is that they only provide static RNA expression pro- and have become a common advanced tool in scRNA-
files. In reality, cells are constantly undergoing dynamic seq analysis. The structure of dynamic processes can be
changes, whether during development and differentia- linear, nonlinear, or branching. Commonly used software
tion, disease progression, and pre- and post-treatment, includes Monocle [14], RNA velocity [26], Palantir [27],
etc. Frequently, scRNA-seq is conducted at various time CytoTRACE [28], and others.
points to gain valuable insights into the development or Monocle is an unsupervised algorithm designed for
response process [23–25]. Time-resolved scRNA-seq is pseudotime inference analysis, capitalizing on the high
primarily studied by experimental approaches or compu- variability in gene expression levels. The latest version,
tational tools, allowing for the inference or acquisition of Monocle3, utilizes UMAP for trajectory inference [14].
dynamic data (Fig. 3A). This enables the study of time- Entropy-based pseudotime analysis is also a method.
resolved scRNA-seq. It leverages the concept that entropy is negatively cor-
related with cell differentiation states. Higher entropy
Computational method values suggest a greater stemness, indicating a more
Computational tools can endow scRNA-seq data, which primitive and undifferentiated state [29].
capture only a static snapshot at a time, with inferred RNA velocity is a prevalent technology in scRNA-
temporal information without resorting to any experi- seq analysis [26]. It operates on the principle that, dur-
mental technologies. These approaches are commonly ing dynamic regulatory processes, unspliced (nascent)
referred to as pseudotime analysis. Pseudotime analy- mRNA always appears before spliced (mature) mRNA.
sis, also known as trajectory inference, ranks potential By assessing the abundance of both unspliced and spliced
dynamic processes in cells based on the heterogeneity mRNA, one can reveal indicators of dynamic changes in
of transcriptional expression levels. These approaches the transcriptome over time.

Fig. 3 The principles of key technologies in the dimensions of (A) time and (B) space at the single-cell level
Wu et al. Biomarker Research (2024) 12:110 Page 6 of 28

Palantir is a pseudo-time algorithm based on stochas- core feature of transcription dynamics at the single-cell
tic processes that, through dimensionality reduction and level. To address the constraint prohibiting large-scale
manifold analysis, effectively captures the continuity of scSLAM-seq, researchers developed new approaches that
cell states and models the transition from low-differen- integrate high-throughput unique molecular identifier
tiation cells to terminally differentiated cells [27]. It can (UMI)-based scRNA-seq analysis with metabolic label-
be applied to a variety of tissue types and is particularly ing. It has successfully enabled the acquisition of new/
well-suited for addressing less-studied differentiation old transcriptomes from thousands of single cells. It has
systems. been applied in transcription factor activity and cell state
CytoTRACE leverages gene counting and expression trajectories during neuronal activation [33], as well as
to reconstruct cell trajectories, enabling the prediction of the transcriptional dynamics in colorectal cancer cells
relative differentiation states of single cells based on sin- treated with DNA-demethylating drugs [34]. We antici-
gle-cell RNA expression data, without being constrained pate the potential of dynamic transcriptomics application
by specific time scales or the presence of continuous in response to external stimuli (including viral infections
developmental processes in the data [28]. Additionally, and pharmacological interventions), embryonic devel-
CytoTRACE is independent of tissue type, species, and opment, cell differentiation, tumor progression, and
platform and can be used to predict differentiation status immune cell transformation is immense. So far, the appli-
in scRNA-seq data without any prior information. cation of these technologies in vivo remains unexplored,
despite their established utility at the bulk level [35]. It
Experimental method should be emphasized that the combination of scRNA-
As mentioned above, trajectory inference for single- seq with metabolic labeling in vivo is a feasible avenue
cell analysis provides an avenue to explore the temporal that warrants further identification.
information about cells. The fundamental limitation is In addition to 4sU, Battich et al. presented an approach
that it is only a computational inference and cannot rep- that utilizes 5-ethynyl-uridine (EU) and click chemistry
resent the actual existence of dynamic RNA processes. to separate new and old RNA, thus providing a dynamic
Emerging experimental technologies allow us to distin- view of RNA synthesis and turnover [36]. 5-Ethynyl-
guish time-resolved phenomena in reality by chemical or 2-deoxyuridine (EdU), a thymidine analog, was applied
biological methods (Table 1). for scRNA-seq and scATAC-seq dynamics [37]. Meta-
bolic labeling-based RNA velocity can accurately reca-
Metabolic RNA labeling pitulate the dynamics of gene expression.
Metabolic RNA labeling effectively integrates with high-
throughput scRNA-seq, addressing limitations pertaining Fluorescent timer
to RNA transcription dynamics [8]. The technology was The research by Gehart et al. showcased that using fluo-
initially applied at the bulk RNA level [30]. Currently, the rescent reporters offers a promising way to reveal real-
most typical metabolic RNA labeling in scRNA-seq is a time-resolved scRNA-seq [38]. Neurog3 is transiently
nucleoside analog called 4-thiouridine (4sU) [31, 32]. The expressed in enteroendocrine (EE) progenitor cells.
brief technical principle is as follows: After tissue dissoci- They engineered the integration of three independent
ation, the culture medium is supplementary with 4sU. In proteins: NEUROG3, dTomato (red), and destabilized
the process of nascent RNA synthesis, 4sU replaces uracil mNeonGreen (green) to study the dynamics of EE cell
(U). Reverse transcriptase misread 4sU as cytosine (C). development. The differing decay rates of mNeonGreen
This misreading leads to incorrect pairing, where adenine and dTomato enable the measurement of the actual time
(A), which normally pairs with U, is replaced by guanine elapsed since Neurog3 expression in individual cells,
(G). This ultimately results in T-to-C substitutions in the determined by the red: green fluorescence ratio. Integrat-
labeled new RNA. ing this approach with scRNA-seq, they have successfully
Single-cell, thiol-(SH)-linked alkylation of RNA for created a real-time-resolved map, revealing the intri-
metabolic labelling sequencing (scSLAM-seq) pioneered cate process of EE cell differentiation and development.
the application of the metabolic labeling method at the Recently, Kirschenbaum et al. developed fluorescent-
single-cell level [32]. This breakthrough identified DEGs based dynamics scRNA-seq called Zmen-seq in vivo,
in mouse fibroblasts infected and uninfected with cyto- which uncovered immune dysfunctional trajectories of
megalovirus (CMV) by distinguishing between total, old, glioblastoma [39].
and new RNA matrices. The study highlighted a critical
insight: most DEGs are primarily detectable in new RNA, CRISPR/Cas9
eluding detection in analyses of old or total RNA. Addi- Lineage tracing can be deduced by the patterns of muta-
tionally, it revealed that interferon and NF-κB varied sig- tions shared between cells. The emergence of CRISPR/
nificantly with different levels of infection, which was a Cas9 technology facilitates the deliberate introduction
Table 1 Time-resolved single-cell technologies based on experimental method
Experimental Pub- Principle Advantages and disadvantages Cultivation Research object Number of Ref-
Method lished method cells er-
Wu et al. Biomarker Research

date ence
scSLAM-seq 2019 Jul 4sU Advantages: captures transient and dynamic nature of transcription; applicable for studying single- In vitro lytic mouse 110 [32]
cell response to perturbations cytomegalovirus
Disadvantages: single cell sample size is too small; extracting mRNA from cells to perform chemical infection
conversion
NASC-seq 2019 Jul 4sU Advantages: identifying newly synthesized RNA In vitro K562 and Jurkat < 200 [44]
(2024) 12:110

Disadvantages: single cell sample size is too small; extracting mRNA from cells to perform chemical cells
conversion; restricted to cell populations
Sci-fate 2020 4sU Advantages: using combinatorial indexing to detect more cells; a greater number of genes detected In vitro Cell cycle and > 6,000 [45]
Aug per cell; in situ 4sU (high reaction efficiency and low mRNA loss) glucocorticoid
Disadvantages: requires complex experimental setups, leading to cell loss receptor activation
scNT-seq 2020 4sU Advantages: droplet microfluidics, high-throughput and UMI-based In vitro Neuronal activation ∼ 55,000 [33]
Oct Disadvantages: limited sequencing coverage for scRNA-seq; cannot wash away cell-free RNAs in the
droplets
Well-TEMP-seq 2023 4sU Advantages: low cell loss rate, high-throughput and cost-effective; In vitro Anticancer drugs 16,000 [34]
Mar Disadvantages: only for in vitro experiment treat colorectal
cancer cells
scEU-seq 2020 EU Advantages: high UMI; high-throughput In vitro Intestinal organoids 5,422 [36]
Mar Disadvantages: requires specialized reagents and procedures; physical separation of new and old
transcripts to conduct two libraries
TrackerSci 2023 EdU Advantages: identifying newborn brain cells; revealing cell dynamics In vitro/In brain newborn cells ∼ 800,000 [37]
Seq Disadvantages: a lesser detection of the quiescent cell population vivo
Fluorescent timer 2019 dTomato (red); Advantages: offers real-time tracking of gene expression changes during the biological process; In vitro/in Mice and organoids / [38]
Feb destabilized from instantaneous changes to long-term process vivo
mNeonGreen
(green)
Zmen-seq 2024 fluorophore Advantages: exploring biological processes in vivo for multiple days; In vivo Immune cells in 8,976 [39]
Jan pulse labels Disadvantages: the number of fluorescent timers is limited tumor environment
(TME)
CARLIN 2020 CRISPR-Cas9 Advantages: Combines CRISPR-Cas9 with scRNA-seq for lineage tracing In vivo Genetic lineage / [41]
Jun tracing
Page 7 of 28
Wu et al. Biomarker Research (2024) 12:110 Page 8 of 28

of mutations using guide RNAs (gRNAs) [40]. These technologies into three classes (Table 2) and present the
induced mutations can be applied to readout of lin- primary technologies and principles (Fig. 3B).
eage histories at the single-cell level [41]. This advanced
approach is used to trace cell lineage dynamics during Image-based in situ technologies
embryonic development and reconstruction [42], as well The image-based in situ technologies evolve from in
as to track the growth and dissemination of lung tumor situ hybridization (ISH) and in situ sequencing (ISS).
xenografts in mice [43]. ISH employs labeled probes to hybridize with target
sequences in cells, thereby visualizing the locations of
Spatial information the sequences [49]. ISH-based technologies use com-
Despite significant advancements in scRNA-seq, current plementary fluorescent probes to hybridize with target
scRNA-seq technologies require isolating cells from the sequences and detect them. Single-molecule fluorescence
tissues, resulting in the loss of spatial information, which ISH (smFISH) is a high-resolution technology that uti-
is crucial for investigating intercellular interactions and lizes multiple short oligonucleotide probes coupled with
functional relevance [46, 47]. In recent years, thriving fluorescent moieties to selectively detect transcripts
spatial omics technologies have provided robust tools [50], but it is limited by the throughput of detection.
for analyzing spatial information of cells, with spatially The emerging multiplexed FISH technologies address
resolved transcriptomics acknowledged as Method of the limitation. Sequential FISH (seqFISH) barcodes the
the Year in 2020 [48]. Here, we categorize spatial omics transcripts fixed within cells through multiple rounds of
hybridization, imaging, and probe removal, allowing for

Table 2 Primary spatial transcriptomics and spatial multi-omics technologies

Technology Resolution Single-cell? Data type detected Sample type Genes/transcripts Refer-
detected ence
ISH-based
smFISH Subcellular Yes mRNA Cell 2 genes [50]
seqFISH Subcellular Yes mRNA Cell 12 genes [51]
seqFISH+ Subcellular Yes mRNA Cell 10,000 genes [53]
MERFISH Subcellular Yes mRNA Cell, tissue 140genes, 1,001 genes [54]
section
ISS-based
ISS Subcellular Yes mRNA Cell, tissue 31 transcripts [55]
section
FISSEQ Subcellular Yes mRNA Cell, tissue 8,102 genes [56]
section
STARmap Subcellular Yes mRNA Tissue section 160 ∼ 1,020 genes [57]
STARmap PLUS Subcellular Yes mRNA, protein Tissue section > 20,000 genes [58]
ROI selection-based
Geo-seq Single-cell Yes mRNA FF > 80,00 genes [66]
DSP 10 μm No mRNA, protein FF, FFPE 96 genes, 1,412 genes [68]
Spatial barcode-based
ST 100 μm No mRNA FF Whole transcriptome [72]
10× Visium 55 μm No mRNA FF, FFPE Whole transcriptome [74]
Slide-seq 10 μm No mRNA FF Whole transcriptome [79]
Slide-seqV2 10 μm No mRNA FF Whole transcriptome [80]
HDST 2 μm No mRNA FF Whole transcriptome [81]
Stereo-seq 0.22 μm Yes mRNA FF Whole transcriptome [82]
Decoder-seq 10 ∼ 50 μm No mRNA FF Whole transcriptome [83]
DBiT-seq 10 ∼ 50 μm No mRNA, protein FF, FFPE Whole transcriptome [85]
spatial CITE-seq 10 ∼ 50 μm No mRNA, epitope FF Whole transcriptome [87]
spatial-ATAC-RNA-seq 10 ∼ 50 μm No Chromatin accessibil- FF Whole transcriptome [86]
ity, mRNA
spatial-CUT&Tag-RNA-seq 10 ∼ 50 μm No Histone modifica- FF Whole transcriptome [86]
tions, mRNA
Slide-tags < 10 μm No mRNA, Chromatin FF Whole transcriptome [88]
accessibility, TCR
FF, fresh frozen; FFPE, formalin-fixed paraffin-embedded
Wu et al. Biomarker Research (2024) 12:110 Page 9 of 28

the detection of the entire transcriptome through four sections for subsequent analysis. Laser capture micro-
dyes and eight rounds of hybridization [51]. Nevertheless, dissection (LCM) is a typical technology capable of cap-
it suffers optical crowding when profiling an excessive turing ROIs at resolutions ranging from cell-population
number of transcripts [52]. seq-FISH + is an improved to single-cell via laser cutting [65]. Geographical posi-
seqFISH, profiling 10,000 genes within individual cells by tion sequencing (Geo-seq), an integration of LCM and
expanding the barcode base palette to 60 pseudo colors scRNA-seq, enables the construction of 3D transcrip-
[53]. In addition, it only labels a fraction of transcripts tome atlases, thereby revealing cellular heterogeneity and
during each hybridization cycle to avoid optical crowd- spatial disparities [66]. Additionally, the integration of
ing. In another strategy, multiplexed error-robust FISH high-content imaging, LCM, and multiplexed mass spec-
(MERFISH) can simultaneously image 100 to 1,000 kinds trometry can extend single-cell proteomics to intact tis-
of RNA within individual cells by assigning combinatorial sue, significantly improving biological insight [67]. These
FISH labeling to RNA, followed by successive hybridiza- studies suggest that LCM is a promising technology for
tion and imaging [54]. isolating ROIs within tissues for multi-omics analysis.
ISS, first reported in 2013, utilized padlock probes to GeoMx digital spatial profiler (DSP), a commercially
bind with cDNA produced by reverse transcription, available technology, is capable of spatially profiling RNA
followed by rolling-circle amplification (RCA) to pro- or proteins within the ROIs [68]. It uses a photocleavable
duce rolling-circle products (RCP), which were in situ (PC) linker to connect oligo sequence (DSP barcodes)
sequenced and imaged within tissues or cells [55]. Cur- with RNA probe or antibody in fixed tissue, selecting
rently, fluorescent in situ sequencing (FISSEQ) and ROIs, and releasing barcodes with UV for sequencing.
spatially-resolved transcript amplicon readout mapping This technology has been used to dissect the heterogene-
(STARmap) are two representatives of ISS-based tech- ity of glomerular transcriptional profiler missed by LCM
nologies. FISSEQ is an untargeted strategy that uses in collapsing glomerulopathy [69], as well as to identify
hexamer primers to reverse transcribe RNA in fixed biomarkers associated with immune checkpoint inhibitor
cells, followed by cDNA circularization, and generates (ICI) resistance in non-small cell lung cancer (NSCLC)
a sequencing library, further improving the detection [70]. However, these technologies are limited by through-
throughput of ISS [56]. STARmap hybridizes directly to put, as each ROI requires individual collection and
mRNA with a SNAIL probe and then undergoes RCA to processing.
obtain DNA amplicons, thereby avoiding reverse tran-
scription [57]. The distinctive feature is its capability to Spatial barcode-based technologies
achieve in situ sequencing in three-dimensional (3D) The core of spatial barcode-based technologies is cap-
intact tissue. STARmap PLUS further improves detection turing transcripts within tissues or cells using spatial
throughput and is compatible with both transcriptome barcodes (DNA oligos) array on glass slides [71]. Sub-
sequencing and protein detection within the same tissue sequently, library preparation and next-generation
section, providing a more comprehensive insight into the sequencing (NGS) are performed.
biological systems [58]. Spatial transcriptomics (ST) was a milestone achieve-
The image-based in situ technologies can achieve sub- ment, carrying epochal significance for spatially resolved
cellular resolution [59]. When combined with single-cell transcriptomics [72]. It innovatively captured polyad-
omics technologies, they hold great potential for diverse enylated RNA on spot-equipped slides, with each spot
applications, particularly in neuroscience. For instance, containing unique spatial barcodes, ensuring that each
Shi et al. employed STARmap PLUS on 20 central ner- transcript was precisely mapped back to its respec-
vous system (CNS) tissue sections and integrated the tive spot through the spatial barcode [73]. ST was first
resulting data with the published scRNA-seq atlas, applied in adult mouse olfactory bulb and human breast
achieving molecular cell typing of the CNS [60]. Yao et cancer, achieving RNA sequencing while preserving two-
al. integrated MERFISH and scRNA-seq to construct a dimensional spatial information. 10× Genomic improved
transcriptomic and spatial atlas of the mouse whole brain it and released 10× Visium, which possessed a higher
and built a platform to visualize these data, providing spatial resolution [74]. It has been commercialized and
precious resources for deciphering the complexity of the extensively employed across diverse fields such as devel-
mammalian brain [61]. Additionally, the combinations opmental biology [75], cancer biology [76], as well as
can be applied in hematology [62], stem cell research neuroscience [77, 78]. For instance, Olaniru et al. applied
[63], and developmental biology [64]. the integration of scRNA-seq with 10× Visium to the
developing human fetal pancreases, analyzing the differ-
ROI selection-based technologies entiation and maturation processes of various cell types
These technologies employ specific methodologies to [75]. Galeano Niño et al. applied 10× Visium to oral squa-
precisely select regions of interest (ROIs) from tissue mous cell carcinoma and colorectal cancer, identifying
Wu et al. Biomarker Research (2024) 12:110 Page 10 of 28

the identity and in situ position of the microbial commu- co-mapping transcriptome and epitope. A very recently
nities within the tumors [76]. Hasel et al. integrated bulk developed technology, Slide-tags, can label single nuclei
RNA-seq, and scRNA-seq with 10× Visium, uncovering with spatial barcodes and isolate them for multi-omics
spatiotemporal heterogeneity in the response of different analysis [88]. Altogether, these spatial multi-omics tech-
astrocyte subsets to inflammation in the brain [78]. These nologies provide more comprehensive biological infor-
applications suggest that 10× Visium is a highly promis- mation, deepening our understanding of the intricate
ing technology for dissecting spatial information. spatial and molecular interactions in the field of life sci-
Slide-seq captures transcripts on a slide equipped ence and biomedical research.
with random 10-µm DNA-barcoded beads and gains the
positions of barcodes via in situ indexing, enabling spa- Genome
tial transcriptomic analysis at a near-cellular resolution Although genomes are generally considered to be sta-
[79]. Stickels et al. optimized Slide-seq and reported the ble, there is still a small possibility of genetic mutations
highly sensitive Slide-seqV2, leading to a tenfold increase occurring with each DNA replication. Single-cell whole-
in RNA capture efficiency [80]. High-definition spatial genome sequencing (scWGS) is capable of elucidating
transcriptomics (HDST) utilizes a split-pool approach genomic heterogeneity and can therefore be utilized to
to generate a dense and barcoded bead array, capturing analyze genomic mutations in single cells. This technol-
RNA at a 2-µm resolution [81]. Recently developed spa- ogy involves the isolation of individual cells or nuclei fol-
tial enhanced resolution omics-sequencing (Stereo-seq) lowed by whole-genome amplification (WGA), library
utilized a randomly barcoded DNA nanoball patterned preparation, and sequencing [89]. It has been applied to
array chip to achieve single-cell resolution and has been uncover somatic mutations in multiple cell types, such
employed to delineate the spatiotemporal transcriptomic as human bronchial epithelial cells [90], and B lympho-
landscape of mouse organ development [82]. cytes [91], offering novel insights into the pathogenesis of
However, the aforementioned spatial barcode-based diseases.
technologies suffer several constraints, such as multi- Single nucleotide polymorphism (SNP), a common
cellular resolution, low sensitivity, and the necessity for form of genetic variation, is caused by the transition,
sequencing to obtain positional indexing. To address transversion, insertion, or deletion of individual bases.
these constraints, Cao et al. presented a dendrimeric Expression quantitative trait loci (eQTL) analysis can
DNA coordinate barcoding design for spatial RNA profile genetic variations (especially SNP) that affect
sequencing (Decoder-seq) [83]. The central innovation gene expression, offering enhanced insights into the
was to employ a microfluidics-assisted combinational relationship between genetic variants and gene regula-
barcoding approach to create high-density spatial bar- tion [92, 93]. The integration of scRNA-seq with eQTL
code arrays on a 3D dendrimeric nanosubstrate, enabling was first reported in 2018 [94]. Kang et al. utilized mul-
cost-effective, highly sensitive, near-cellular resolution tiplexed droplet scRNA-seq to profile eight immune cell
spatial transcriptomics research. They utilized it to spa- populations from 23 donors and subsequently conducted
tially resolve the distribution of low-expressed olfactory eQTL analysis, identifying 32 cis-eQTLs, 22 of which
receptor genes and accurately depict the spatial single- were cell-specific. Recently, Ding et al. constructed the
cell landscape of the hippocampus. first integrated human sceQTL database, which com-
Recently, spatially resolved multi-omics has been rec- prises ∼ 16 million SNPs and ∼ 0.69 million sceQTLs [95],
ognized as one of the noteworthy technologies in 2023 providing a valuable resource for disease susceptibility
[84]. Fan’s group pioneered spatial multi-omics technol- gene discovery.
ogy. They innovatively employed two orthogonal chips In addition, multiple multi-omics technologies for inte-
equipped with parallel microfluidic channels to deliver grative analysis of genome and transcriptome have been
DNA barcodes to tissue sections, developing the first developed, among which genome and transcriptome
spatial multi-omics technology, deterministic barcod- sequencing (G&T-seq) [96] and gDNA-mRNA sequenc-
ing in tissue for spatial omics sequencing (DBiT-seq), ing (DR-seq) [97] stand out as two representative tech-
enabling simultaneous detection of the whole tran- nologies. G&T-seq utilizes oligo-dT-coated beads to
scriptome and 22 proteins [85]. Furthermore, the group isolate mRNA and DNA, which are subsequently ampli-
extended this approach to spatial ATAC-RNA-seq and fied and subjected to whole transcriptome sequencing
spatial assay of cleavage under targets and tagmentation and WGS, respectively. Whereas DR-seq employs a pre-
and RNA sequencing (spatial CUT&Tag-RNA-seq) [86], amplification-and-separation strategy to decouple DNA
which achieve co-profile of epigenome and transcrip- and mRNA molecular analytes, thereby avoiding physical
tome, as well as spatial co-indexing of transcriptomes nucleic acid separation before amplification. Therefore,
and epitopes for multi-omics mapping by highly parallel compared with G&T-seq, DR-seq has a lower cross-con-
sequencing (spatial CITE-seq) [87], which is capable of tamination rate and a higher recovery rate. Collectively,
Wu et al. Biomarker Research (2024) 12:110 Page 11 of 28

these technologies hold vast potential for deciphering methods of scATAC-seq were developed in 2015, which
genetic variations and their impacts on gene expression. enabled the exploration of chromatin accessibility at the
single-cell level. The first one utilized a programmable
Epigenome microfluidics platform to capture single cells, followed
Epigenomics aims to explore how chemical modifica- by Tn5 transposase tagmentation and library amplifica-
tions and spatial structure alterations of the genome tion with cell-identifying barcoded primes [111]. The
affect gene function and expression regulation. Decipher- other one utilized an integrative method combining com-
ing the epigenomic features such as DNA methylation, binatorial cell indexing with ATAC-seq to analyze chro-
chromatin accessibility and histone modifications at the matin accessibility within over 15,000 cells [112]. 10×
single-cell level allows us to study cell lineages and differ- Genomics developed a Chromium platform and applied
entiation states [98]. it to scATAC-seq, which combined Tn5 transposase tag-
In eukaryotes, the most common DNA methylation mentation within bulk nuclei and single-nuclei isolation
occurs on the fifth carbon atom of C within the CpG through the droplet system [113]. Currently, the integra-
island, yielding 5-methylcytosine (5-mC). Bisulfite con- tion of scRNA-seq and scATAC-seq has been applied to
version is the gold standard for DNA methylation anal- explore the regulation of human developmental hemato-
ysis [99]. The primary principle is that methylated C in poiesis [114], as well as to find potential therapeutic tar-
DNA remains unchanged after treatment with bisulfite, gets for clear cell renal cell carcinoma [115].
whereas unmethylated C is converted to U [100]. After Histone modifications are chemical modifications that
PCR amplification and high-throughput sequencing, the occur at specific sites on histone molecules, thereby
bases that methylated can be ascertained by compari- affecting chromatin structure stability and gene expres-
son with reference sequences. Single-cell DNA methyla- sion regulation [116]. Chromatin immunoprecipitation
tion sequencing based on bisulfite conversion and NGS sequencing (ChIP-seq) is a common method to pro-
can be categorized into single-cell reduced representa- file histone modifications [117]. Single-cell ChIP-seq
tion bisulfite sequencing (scRRBS) [101] and single-cell (scChIP-seq) tags nucleosomes with barcodes via a drop-
whole-genome bisulfite sequencing (scWGBS) [102], let microfluidic platform before conventional ChIP-seq,
which achieve single-base resolution. These technologies and it was employed to interrogate the chromatin land-
play crucial roles in investigating cell differentiation and scapes of breast cancer [118]. However, ChIP-seq has a
development. The first single-cell multi-omics technology high demand for experimental samples. To address the
achieving co-profile of the DNA methylome and tran- obstacle, Kaya-Okur et al. introduced CUT&Tag, which
scriptome is single-cell methylome and transcriptome utilizes Protein A-Tn5 transposase to cleave the DNA
sequencing (scM&T-seq) [103], which utilizes G&T-seq sequences bound by targeted protein and integrates
to separate and amplify genomic DNA and mRNA from sequencing adapters with the cleaved sequences [119].
the same single cell and applies scBS-seq [104] to gener- On this basis, single-cell CUT&Tag (scCUT&Tag) inte-
ate DNA methylation data. In addition, single-cell triple grated CUT&Tag with 10× Genomics scATAC-seq pro-
omics sequencing (scTrio-seq), which co-profiles the tocol, enabling the investigation of histone modifications
genome, DNA methylome, and transcriptome through at single-cell resolution [120]. It was applied to explore
scRRBS and WGS, has been applied to 25 cancer cells the histone modification features of regulatory elements
and identified two distinct subsets [105]. However, one and gene bodies in the central nervous system cells of
constraint of these technologies is that bisulfite conver- mice. Recently, some multi-omics technologies have
sion involves intense chemical reactions that can lead been developed, such as Paired-Tag [121] and combined
to significant DNA degradation and consequent loss of assay of transcriptome and enriched chromatin binding
information. (CoTECH) [122], which utilize a combinatorial barcod-
Chromatin accessibility, a key epigenetic feature, plays ing strategy, achieving co-profile of histone modifications
a pivotal role in regulating gene expression by allowing and transcriptome in single cells.
transcriptional machinery to interact with regulatory ele-
ments, thereby facilitating the initiation or suppression Cellular protein and epitope
of gene transcription in open chromatin regions [106]. Single-cell proteomics is a more nascent field. Tran-
Recently a variety of technologies have been developed scriptomic features may not exhibit a comprehensive
to interrogate chromatin accessibility [107–109], whereas snapshot of cellular heterogeneity since similar gene
ATAC-seq barges to the forefront as a landmark break- expression profiles may be identifiable in other modali-
through that utilizes the Tn5 transposase to fragment ties that are simultaneously measured. Indeed, the tran-
open chromatin and labels the genome with sequencing scriptomes and proteomes represent distinct molecular
adaptors [110]. Subsequently, the labeled genome under- modalities, such as post-translational modifications that
goes PCR amplification and sequencing (Fig. 2F). Two cannot be captured by transcriptomics. It’s crucial to
Wu et al. Biomarker Research (2024) 12:110 Page 12 of 28

simultaneously identify the transcriptome and protein Immune Repertoire

abundance at the single-cell level. Mass spectrometry- Immune repertoire (IR) encompasses the diversity of T/B
based single-cell proteomics (scMS), achieving a detec- cells in a particular environment, indicative of the capac-
tion depth of about 1,500 ∼ 2,500 proteins, is the most ity of the immune system to respond to external stimuli at
successful and extensively discussed in this review [123]. a particular moment. T/B cells, as the primary cell popu-
Furthermore, imaging-based approaches address the lations in the specific immune system, are at central focus
issue of spatial distribution [123]. Integrated with the in immunological research, where deciphering their char-
burgeoning scRNA-seq technology, spatial resolution acteristics and functions remains key to delineating the
scMS was applied to explore pivotal modalities of the immune microenvironment. scTCR-seq and scBCR-seq
skin dermal fibroblast cells [124]. The challenge of quan- enable the determination of the full-length or comple-
tifying proteins in sequencing is addressed by leveraging mentarity-determining region 3 (CDR3) gene sequences
the binding of specific antibodies linked to oligonucle- related to TCR and BCR, respectively, at the single-cell
otide for translation and amplification, thus overcoming level. It complements the limitation that scRNA-seq only
the limitations of existing RNA-seq methods, which are obtains the transcriptome landscape, providing deep
unable to directly measure proteins. Cellular indexing of insights into the behavior of T/B cell populations, and
transcriptomes and epitopes by sequencing (CITE-seq), enabling researchers to understand and possibly manipu-
one of the most widely used methods that sequence cellu- late these responses for therapeutic purposes. The main
lar surface protein abundance via oligonucleotide-conju- directions of application are TME research, the evalua-
gated antibodies, combining protein-level and RNA-level tion and monitoring of immune/infectious diseases, and
insights within single-cell analyses (Fig. 2C) [125, 126]. TCR, BCR, and antibody screening.
Furthermore, they developed the applicable strategy of
integrated multi-omics single-cell data to adjust for the TCR
varying multi-omics quantifications. They constructed a It is well-known that T cells recognize the peptide-loaded
comprehensive atlas on the circulating human immune major histocompatibility complex (pMHC) presented by
system based on the multimodal definition [7]. Trzu- antigen-presenting cells via the TCRs, thereby trigger-
pek et al. discovered a novel subset of T regulatory cells ing the subsequent immune response to kill cancerous
(Tregs) with significantly upregulated CD80 and CD86, or infectious cells [132]. Deciphering TCR repertoire in
as revealed by antibodies-based sequencing. Moreover, varying situations is the foundation for understanding
their data indicated a low correlation between RNA and mechanisms, diagnostics, and developing new vaccines.
protein levels [127]. TCR is composed of α chain (TRA) and β chain (TRB),
Proteins and other epitopes in cells are constantly which are produced by combinatorial rearrangement of
dynamic changes in spatiotemporal processes, thus add- gene segments: variable (V), diversity (D) (exclusive to the
ing information about proteins or other epitopes to β chain), joining (J), and constant (C). This recombina-
scRNA-seq can accelerate the understanding of cellular tion process results in a vast diversity of TCR repertoire,
states and functions. Recently, Glycan epitopes on the predominantly determined by the hypervariable CDR3
surface have been described as the specific cell states and [133]. Given that the probability of identical rearrange-
types, some studies have also focused on it [128–130]. ments occurring in the absence of selection pressure is
SUrface-protein Glycan And RNA-seq (SUGAR-seq) extremely low, TCR sequencing has been identified as a
revealed unique surface glycan profiles in tumor-infil- valuable indicator for antigen-driven clonal expansion,
trating lymphocytes (TILs) [128]. Yu et al. have revealed reflecting antigen specificity and response.
that N-acetyllactosamine (LacNAc), a glycans related to The history of high-throughput TCR sequencing has
immune receptor signaling, serves as a distinct indicator been introduced thoroughly in this review [134]. The
for discerning the glycolytic activity and effector function most widespread application involves simultaneous
of CD8+ T cells [129]. scRNA-seq and scTCR-seq. In this method, cDNA is
A series of research has proved that cellular epitope partly used for the enrichment of TCR, while the other
information can reveal the phenotypes that could not be is utilized to construct gene profiles (Fig. 2A). This tech-
detected by scRNA-seq alone. Although existing technol- nology has been extensively commercially adopted by
ogies empowered scRNA-seq analysis by providing rich multiple companies. In RNA, TRA, and TRB sequencing,
surface protein and epitope resources, it seems difficult sequences shared with the same barcode are computa-
to establish high-throughput single-cell proteomics in tionally inferred as a pair. However, a current limitation
parallel [131]. of this technology is the possibility of missing a chain
and incorrectly matching with more than two TCR
sequences.
Wu et al. Biomarker Research (2024) 12:110 Page 13 of 28

A series of heavyweight studies on the TILs have exhib- [145], conducting vaccine studies [146], etc., and proved
ited the T cell and TCR clonotype diversity, constructing invaluable in studies investigating the dynamics of the
the landscape of T cell heterogeneity and dynamics in the immune system. From a clinical perspective, it helps
TME [135–137]. diagnose and monitor tumor immunology [144], infec-
In routine analysis, the primary objective is to map tious diseases [145, 147], autoimmune diseases, and
and analyze the diversity of clonotypes or CDR3. Addi- immunodeficiencies.
tionally, clonotype overlap represents another critical
dimension, empowering the characterization of TCR Microbiome
sequences shared across different tissues or tracking of Microbiome refers to the collection of microorganisms
key clonotypes during therapeutic interventions. From living in a particular environment. The main technolo-
the excellent work of Zhang’s group, they pioneered the gies in the microbiome field are metagenomic sequenc-
development of multiple indicators such as single T cell ing and 16 S rRNA sequencing [148–150], updating our
analysis by RNA sequencing and TCR tracking (STAR- understanding of microbial community on a more micro-
TRAC) (for clonal expansion, tissue migration, and state scopic level (Fig. 2D). Recently an advanced technology
transition), Ro/e, and OR (for tissue distribution) to assess called barcoding bacteria for identification and quanti-
the status of TCR [135–137]. fication (BarBIQ), achieved precise single-base in 16 S
It is crucial to explore the differentiation of TCRs in cell rRNA sequencing through the use of unique barcodes
types/ tissues/ conditions. Chen et al. developed TCRdb, and a droplet-based approach [151]. Microbiomes can
a comprehensive TCR database, that aids in identify- be assessed at the single-cell level, addressing the issue
ing the states and functions of specific sequences [138]. where traditional methods may obscure the simultane-
Another significant challenge is unraveling the binding ous measurement of cell counts for each type of bacteria.
between TCR-pMHC in the context of infectious diseases In the context of scRNA-seq, although it has become a
or tumors. Advanced bioinformatic algorithms accelerate transformative technology for profiling gene expression
research breakthroughs in this field. Among these tools, levels in thousands of eukaryotic cells, challenges such as
NetMHCpan stands out in predicting the binding affin- the low volume of RNA, no polyadenylate tail in bacterial
ity between peptides and MHC-I/II, respectively [139]. RNA, and resistant cell wall have long hindered the adap-
Additionally, Gao et al. have developed a computational tation of scRNA-seq technology to microbes. To over-
tool to predict TCR-peptide binding via a machine learn- come these obstacles, Kuchina et al. introduced microbial
ing method [140]. split-pool ligation transcriptomics (microSPLiT), a tech-
In all, scTCR-seq and associated analyses enhance the nology that can identify the scarce subpopulations of
exploration and application of TCR sequencing. They cells down to a minuscule proportion of 0.142%, which
facilitate the exploration of mechanisms, helping cancer was crucial for revealing rare cellular states that are sig-
early-stage diagnosis, treatment selection, and progno- nificant from a physiological perspective [152]. Similarly,
sis prediction, and designing engineering therapeutic BacDrop was developed for bacterial scRNA-seq, identi-
strategies. fying bacterial types and quantifying the number of spe-
cific types of cells [153]. Besides, Microbe-seq applied
BCR microfluidic-droplet operation and bioinformatic analy-
Similar to TCR, BCR consists of immunoglobulin heavy sis to obtain the genomes of numerous microbes with
chains and light chains​​, the diversity of it is produced by single-cell resolution, and most single-amplified genomes
gene rearrangement of V(D)J gene. Based on its struc- had a purity of over 95% [154].
ture, BCR plays a central role in the adaptive immune The intratumor microbiome has emerged as a novel
response by recognizing specific antigens, which trig- and rapidly evolving research frontier, with the discovery
ger B cell activation, subsequently producing antibod- of microorganisms in various cancer types, including in
ies [141]. Around a decade ago, paired scBCR-seq was some organs traditionally considered to be sterile, pri-
introduced [142, 143]. scBCR-seq allows for a thorough marily in gastrointestinal cancers [155–157]. However,
analysis of BCR repertoires, providing crucial informa- the subtle relationship between them and cancer remains
tion about their diversity and function at the single-cell unclear, sequencing technologies may be an effective
level. More recently, Ian et al. challenged limited infor- solution. Galeano et al. introduced invasion-adhesion-
mation about BCR-seq to antigen specificity and devel- directed expression sequencing (INVADEseq) targeting
oped LInking B-cell Receptor to Antigen specificity a conserved area of the bacterial 16 S rRNA, enabling
through sequencing (LIBRA-seq) to confirm for HIV- the effective creation of cDNA libraries containing bac-
and influenza-specific antibodies [6]. Combined with terial transcripts derived from human cells associated
scRNA-seq, scBCR-seq was applied for producing cell with bacteria. The technology is crucial to uncovering the
atlas [144], identifying different neutralizing antibodies
Wu et al. Biomarker Research (2024) 12:110 Page 14 of 28

complexity of microbiota interactions within tumor tis- for complex systems [173]. This promising technology
sues [158, 159]. has been effectively integrated with RNA-seq and flow
Another interesting bioinformatical approach called cytometry. Its combination with sing-cell technology is
Single-cell Analysis of Host-Microbiome Interactions bound to reflect CCIs from a real perspective and could
(SAHMI) [160], is quite fascinating as it systematically offer real insight into the exploration of tumor-reactive
extracts real microbial signals and quantifies microbiome TCR.
profiles directly from mammalian host sequencing data.
SAHMI advances a similar method into the realm of sin- Perspective in application in biological research
gle-cell analysis, enabling the identification of microbial Currently, single-cell multi-omics technologies are rap-
species related to specific cell types and uncovering the idly evolving, offering robust tools for depicting intricate
relationship between microbial and transcriptome pro- cellular landscapes. They have been applied to a diversity
files, facilitating a deeper investigation into their contri- of fields including, but not limited to, cell atlas construc-
bution to intercellular communication networks [160]. tion, developmental biology, pathways identification, and
novel targets discovery, with remarkable achievements
Metabolome (Fig. 4).
Metabolome focuses on the biochemical reactions within
cells, encompassing the collection of all metabolites of Cell atlases construction
tissue in a specific physiological period and metabolic Constructing cell atlases is a common function of single-
features (Fig. 2E). The field of single-cell metabolo- cell multi-omics technologies. Cell atlases, as one of the
mics marks a pivotal era in unraveling the complexities seven noteworthy technologies in 2024 [175], can display
of cellular processes at the individual cell level. Mass detailed information about various cell types within dif-
spectrometry (MS) [161–164] and nuclear magnetic ferent organisms, allowing for the characterization of cel-
resonance (NMR) spectroscopy [165] are widely utilized lular diversity [176], the analysis of cellular heterogeneity
technologies for metabolomics analysis. Recent advance- [177], as well as the discovery of novel cell types [178].
ments have enabled the analysis of single-cell metabo- In recent years, the great advances in single-cell multi-
lomics and even at the level of single-organelle [166]. A omics technologies have enabled scientists to character-
comprehensive overview of the history and advances of ize various molecular information within individual cells,
metabolomic methodologies is provided in this review deepening our understanding of different cell types.
[167], but this review refrains from further elaboration. The largest cell-atlas initiative, the Human Cell Atlas
Importantly, it remains a challenge to combine metabo- was launched in 2017, aiming to integrate single-cell
lomics with other single-cell multi-omics technolo- omic data into comprehensive atlases, thereby enhancing
gies given the limited sample size of single cells and the our understanding of cell development, physiology, and
destructive quality of analyses and sequencing. In this CCIs [74]. Currently, with the joint efforts of scientists,
respect, advanced technologies that possess the poten- cell atlases of a wide range of organs and disease tissues
tial to integrate with other omics-based technologies are have been constructed [179, 180]. By comparing the cell
warranted. atlases of healthy and diseased states, we can uncover
the mechanisms underlying diseases and advance their
Cell-cell interactions (CCIs) diagnosis and treatment [181]. In addition, single-cell
With the advancement of bioinformatic analysis, tools multi-omics technologies can be applied to construct cell
developed based on scRNA-seq for inferring cell-cell atlases of organoids for fundamental research as dem-
communications, such as CellChat [168], CellPhoneDB onstrated in research that constructed a human brain
[13], NicheNet [169], and CellCall [170], have become organoid development atlas by co-profiling of transcrip-
mainstreams. However, these methods of inferring CCIs tome and chromatin accessibility to investigate the regu-
through algorithms are influenced by parameters and do lation of cell fate decisions [182]. Therefore, constructing
not necessarily represent actual occurrences. The appli- comprehensive cell atlases with single-cell multi-omics
cation of chemical biology tools in medical research is technologies will have profound impacts on biological
expanding, with proximity labeling technologies that may research and human health.
become game changers for reflecting real-world CCIs
[171–174]. A groundbreaking research reported that Developmental biology
FucoID, a chemical biology tool for capturing tumor anti- A principal application of single-cell multi-omics tech-
gen-specific T cells through dendritic cell interactions nologies in developmental biology is lineage tracing,
using fucosyltransferase (FT) [171]. Based on this, they which aims to track the progeny of individual cells to
further developed an advanced platform for T cell-cancer investigate cellular differentiation trajectories [183, 184].
cell and B cell-dendritic cell (DC) interactions adapted Compared with traditional methods to study cell lineage
Wu et al. Biomarker Research (2024) 12:110 Page 15 of 28

Fig. 4 Applications of single-cell multi-omics in biological research and clinical practice

with heritable tags or naturally occurring somatic muta- of human hematopoiesis, revealing the clonal architec-
tions [185], single-cell multi-omics technologies provide ture, functional heterogeneity, and age-related changes
powerful tools for delineating comprehensive lineage of hematopoietic stem cells. Another single-cell lineage-
relationships and diverse cellular states. A newly devel- tracing technology, Camallia-seq, enables integrative
oped multi-omics technology, single-cell Regulatory analysis of chromatin accessibility, DNA methylation,
multi-omics with Deep Mitochondrial mutation profil- transcriptome, as well as cell lineage information, bring-
ing (ReDeeM), utilizes naturally occurring mitochondrial ing new insights into how cell fate decisions are regulated
DNA mutations as barcodes for lineage tracing while and how cell identities are maintained under different
analyzing transcriptome and chromatin accessibility modalities [187].
[186]. It was employed to construct a phylogenetic tree
Wu et al. Biomarker Research (2024) 12:110 Page 16 of 28

Additionally, single-cell multi-omics technologies have comprehensive gene regulatory network and elucidate
been employed for multiple stages of embryonic develop- the regulatory and causal relationships between various
ment, including preimplantation [188, 189], implantation molecules, thus holding great potential in discovering
[190], gastrulation [191], and early organogenesis [192], novel targets [200]. Olatoke et al. performed an integra-
to explore cell fate decisions of embryonic development tive scRNA-seq/single-nucleus ATAC sequencing (snA-
from multiple dimensions, thereby providing a paradigm TAC-seq) analysis on lymphangioleiomyomatosis (LAM)
to decipher the molecular programs of tissue architec- to construct a HOX-PBX gene regulatory network that
ture and cellular organization [193]. To comprehensively controlled the survival of LAM cells, thereby providing
understand embryonic development, it is essential to potential therapeutic targets for LAM [201]. Pozniak et
explore the spatial information of cells, as it is one of al. integrated single-cell transcriptomics with spatial
the key factors determining cellular identity. Integrated transcriptomics and proteomics to investigate mela-
analysis of single-cell and spatial transcriptomics can be noma, revealing a TCF4-dependent regulatory network,
applied to embryonic development, as in a study that which orchestrated multiple transcriptional programs
precisely characterized human embryonic limb develop- leading to immunotherapy resistance [202]. Targeting
ment over time and space [194]. TCF4 can enhance the sensitivity of melanoma to ICI
Altogether, with rapid advances in lineage tracing, sin- and targeted therapy. In another study, scRNA-seq and
gle-cell multi-omics, and spatial transcriptomics, we will ST were applied to characterize the cellular composition
address fundamental questions of developmental biology, and spatial structure of multiple primary lung cancers
achieving a better understanding of cell differentiation (MPLCs), finding that TNFRSF18 was highly expressed
and development. in T&NK cells within tumor tissues [203]. TNFRSF18 has
been demonstrated to be associated with non-response
Pathways identification to anti-PD-1 therapy in lung cancer [204]. Thus, it is
The currently thriving single-cell multi-omics tech- anticipated that single-cell multi-omics and spatial tech-
nologies are applied to identifying signaling pathways nologies will create a more comprehensive framework,
required for cellular function, shedding light on the providing unprecedented opportunities to discover novel
mechanisms of multiple key pathological processes targets for disease intervention.
[195–199]. Fan et al. performed an integrative analysis
of cervical squamous cell carcinoma (CSCC) utilizing Perspective in applications in clinical practice
scRNA-seq, Stereo-seq, and spatial proteomics, iden- The advent of single-cell multi-omics technologies has
tifying eight meta-programs (MP) [195]. Notably, they added breadth and depth to understanding a battery of
revealed that MP6 tumor cells interact with cancer- complex diseases and their pathology including neuro-
associated fibroblasts (CAFs) to shape an immune exclu- logical disease, immune disorders, oncology, and others.
sionary microenvironment via the FABP5-mediated Within this section, we delve into the applications of sin-
transforming growth factor β (TGFβ) pathway. Han et gle-cell multi-omics across diverse fields, underscoring
al. applied scRNA-seq and scATAC-seq to characterize its transformative impact on clinical practice.
neuroendocrine prostate cancer (NEPC) cells, identify-
ing the KIT pathway, which can be activated by FOXA2 Tumor immunology
to maintain cancer cell proliferation [196]. Inhibition Cancer therapies are continually being developed and
of KIT can be a potential strategy for the treatment of optimized, most of which can remodel the TME. As
NEPC. In addition, integrated analysis of epigenomics knowledge of the immune system improves, new immu-
and transcriptomics was applied to investigate the tran- notherapies, represented by ICIs and adoptive cell ther-
scriptional dynamics of the fibrotic kidney, revealing that apy (ACT), are emerging. The capability of single-cell
the TF Nfix could regulate the expression of the apopto- multi-omics technologies to uncover the intricate inter-
sis-related gene Ifi27 [198]. The Nfix-Ifi27 pathway was actions between the diverse cells and cancer cells sheds
also identified, which can cause kidney fibrosis by pro- light on the heterogeneity and complexity of TME, posi-
moting apoptosis. Therefore, leveraging single-cell multi- tioning them as promising tools in the exploration of
omics technologies to identify signaling pathways offers cancer treatment strategies.
crucial insights into the mechanisms of diseases, opening
up promising avenues for the development of innovative ICIs
therapeutic strategies. How do T cells react during ICI therapy? Paired scRNA-
seq and scTCR-seq might provide deep profiling. One
Novel targets discovery study from ICI therapy for NSCLC, deciphered tumor-
The single-cell multi-omics technologies can inte- specific T cell clonotype feature, regional distribution,
grate information at multiple levels to construct a and temporal persistence during ICI therapy [205].
Wu et al. Biomarker Research (2024) 12:110 Page 17 of 28

Another study from Qiu et al. utilized scRNA-seq, par- rRNA-seq and scRNA-seq analysis have been widely
alleled with scTCR/BCR-seq to elucidate the treatment utilized in microbiota gastric cancer [212], pancreatic
response of Epstein-Barr virus (EBV)-associated gas- injury [213], cholangiocarcinoma [214], etc., explor-
tric cancer. Notably, re-emerged clonotypes in ISG- ing the potential relationship between microbe and host
15+CD8+ T cells after treatment among EBV (+) patients cell types by complementing the composition of micro-
were detected and associated with effector T population bial communities and host cell and genetic informa-
expressing CXCL13 in responsive EBV (+) tumor, indi- tion. A groundbreaking study integrated 16 S rRNA and
cating their significant importance in tumor immunoche- scRNA-seq to reveal that Streptococcus anginosus pro-
motherapy response [25]. David Y. Oh et al. assessed the motes gastric tumorigenesis [215]. Jia et al. integrated
transcriptome characteristics of T cells and paired TCR 16 S rRNA-seq with single-cell transcriptomics, TCR-
from human bladder tumors. Unexpectedly, the typical seq, and ATAC-seq to reveal that IPA activates progen-
CD8+ T cell states were unchanged in tumor and normal itor-exhausted CD8+ T cells through H3K27 acetylation
tissues, while cytotoxic CD4+ T cells showed the oppo- modification [216]. It is worth mentioning that Chai et
site. They also managed to predict the therapeutic effect al. employed the Kraken method [217, 218] to process
of anti-PD-L1 in bladder cancer patients based on CD4 scRNA-seq to obtain the bacterial population corre-
signature score [206]. A study combined scRNA-seq, sponding to specific cell types [214].
TCR-seq, and ATAC-seq for integrated analysis, suggest-
ing that TdLN-TTSM cells are primary memory T cells Infectious diseases
that respond to ICI treatment, representing adoptive In the realm of infectious diseases, the technologies shed
these cells a promising immunotherapy approach [207]. light on host-pathogen dynamics, immune responses, and
advanced therapeutic strategies, especially in COVID-19
ACT [145, 146, 219–223]. Su et al. employed single-cell multi-
With the development of engineered T cells, ACT thera- omics (RNA, CITE, TCR/BCR, etc.) to observe unique
pies, represented by T cell receptor-T cell (TCR-T), Chi- dynamics in the behavior of specific CD8+ T cells during
meric antigens receptor-T cell (CAR-T), and TILs have the recuperation phase from COVID-19, among patients
reshaped the landscape of tumor treatment. Tumor- suffering from gastrointestinal sequelae [219]. Besides,
specific TCRs can recognize tumor-specific antigens, the method is of great significance for the development
which provides a solid foundation for the development of and evaluation of vaccines. Through scRNA/TCR/BCR-
TCR-T therapy. Recent research found CXCL13, CD200, seq, Peng et al. systematically profiled the immune land-
and ENTPD1 as unique markers for tumor antigen-spe- scape after vaccinating lipid nanoparticle-mRNA [146].
cific T cells using scRNA-seq and scTCR-seq. On this A study focused on the breakthrough infection and pan-
basis, developed tumor antigen-specific TCR-T cell ther- variant antivirals, and they successfully identified elite
apies have shown significant therapeutic efficacy in autol- neutralizing antibodies (nAbs) repertoire using scRNA/
ogous patient-derived xenograft (PDX) tumors [208]. BCR-seq of B cells, which showed strong neutralizing
Because of the unclear molecular mechanisms of resis- activity targeting numerous variants [145]. In addition, it
tance to CAR-T therapy in acute lymphoblastic leukemia has been found that the crosstalk of specific T cells and B
(ALL), Bai et al. integrated scRNA-seq and CITE-seq to cells following COVID-19 vaccine treatment [220, 221].
compare responders and CD19-positive relapse patients,
during which they confirmed lack of TH2 functional- Cardiovascular disease
ity might be the cause of relapse in CAR-T treatment An in-depth exploration of cardiac disease using single-
[209]. In addition, the researchers conducted a single-cell cell technologies contributing to predicting disease,
multi-omics (RNA, TCR, and CITE-seq) study in TILs therapeutic target discovery, and stratifying patients
from NSCLC patients to establish a neoantigen-targeted [224–226]. The work from Kanemaru et al. employed
T-cell signature characterized by the frequency of clono- sc-RNA-seq, single-nucleus RNA sequencing (snRNA-
types along with the levels of CD39 protein and CXCL13 seq), snATAC-seq, and spatial transcriptomics, paving
RNA. Utilizing this signature, they were able to detect the way for the anatomy and immunology of the heart
neoantigen-reactive TCRs with a success rate [210]. [227]. Delgobo et al. focused on the transgenic T cell
receptor (TCR-M) cells and myocardial infarction (MI).
Host-microbe interactions Using scRNA/TCR-seq, they elucidated TCR-M cells
A series of thrilling advancements in the interaction expressing Treg markers like Foxp3, Il2ra, and Ctla4
between the human body and microorganisms have fully and suppressed cardiac immune responses post-MI and
illustrated the protective or pathogenic effects of bacte- improved cardiac function [228]. A hypertrophy study
ria, scRNA-seq undoubtedly is the promising method applied multiple-dimensional approaches including epi-
to answer the open questions [211]. Integrated 16 S genetic and morphological analysis to the mechanism of
Wu et al. Biomarker Research (2024) 12:110 Page 18 of 28

pressure overload [229]. In addition, a study employed The challenges of single-cell multi-omics
CyTOF, scRNA-seq, and CITE-seq to decipher the Single-cell multi-omics technologies, widely applied in
immune landscapes in the plaques of atherosclerosis (AS) both biological research and clinical practice, have been
and uncover immune alterations related to clinical car- bolstered by advancements in experimental protocols
diovascular events, suggesting potential avenues for AS and data analysis, as well as by a growing consensus on
treatment [230]. their significance. Despite these improvements, they still
face hurdles that impede their widespread applications.
Neurological and brain disease These challenges and obstacles delineate the future devel-
The high complexity of brain cells requires advanced opment and trajectory of single-cell multi-omics. The fol-
single-cell multi-omics technologies to resolve the basic lowing limitations and issues must be taken into account
gene regulation both in healthy and neuropsychiat- when performing single-cell multi-omics analyses.
ric brain tissues [231]. A 2024 review comprehensively Firstly, the high cost and strict sample requirements
compiled studies on Alzheimer’s disease (AD) using of single-cell technologies discourage many researchers
transcriptomics, metabolomics, and other advanced [131]. Developing high-throughput, cost-effective, sam-
technologies, summarizing mechanisms and targets of ple-friendly, convenient single-cell multi-omics technolo-
sex differences in AD progression [232]. Notaras et al. gies is a crucial issue, particularly for the application in
performed an integrative analysis of transcriptome and clinical practice. The high cost of single-cell multi-omics
proteome on schizophrenia organoids to identify two restricts the measurement of large-scale cohorts, lead-
disease-associated factors (BRN2 and PTN). Both BRN2 ing to data that is more often utilized for discovering
and PTN promoted neurogenesis, while PTN also inhib- new insights rather than for validation, which is com-
ited apoptosis. Besides, the depletion of BRN2 and PTN monly achieved by bulk RNA sequencing [25]. Another
can lead to schizophrenia through different mechanisms challenge is the strict requirement for sample quality.
[233]. In addition, Ji et al. demonstrated that Glutamin- Fresh tissue samples are deemed appropriate for single-
ase 1 deficiency in forebrain neurons can lead to autism cell sequencing, whereas freeze-thaw samples are gen-
spectrum disorder-like behaviors by single-cell multi- erally recommended for snRNA-seq [242, 243]. Besides,
omics analysis [234]. the quantity and viability of the cell suspension are also
As mentioned earlier, the combination of single-cell important factors in obtaining high-quality single-cell
sequencing and spatial transcriptomics holds vast prom- data and detecting all cell types in the tissue.
ise for the study of brain diseases. Li et al. employed The emerging single-cell multi-omics broadens the
scRNA-seq and spatial transcriptomics to identify two multidimensionality beyond transcriptomes and raises
distinct microglial subclusters (ICAM and IPAM). ICAM more profound questions. Developing robust and
was related to ischemia, exhibiting pro-inflammatory advanced computational methods to integrate and ana-
characteristics. In contrast, IPAM, associated with the lyze multi-dimensional single-cell data is a pressing chal-
ischemic penumbra, with inflammation-alleviating and lenge to be addressed for the maturation of single-cell
neuroprotective features. Thus, they reported that target- multi-omics [7]. Importantly, these strategies need to
ing specific microglial subclusters is a promising thera- consolidate the data across diverse dimensions and man-
peutic strategy for ischemic stroke [24]. Similarly, Han age potentially significant differences in individual omics
et al. combined scRNA-seq with spatial transcriptomics data, enhancing the understanding of cellular function
to identify LGALS9-CD44 as a crucial pathway after and state. In some contexts, inconsistencies in informa-
ischemic injury. LGALS9 and CD44 exhibited opposite tion can occur among omics data, although the measure-
effects, in which upregulation of LGALS9 favored recov- ment is conducted simultaneously within the same cells
ery from post-ischemic injury, whereas knockdown of [7, 129].
CD44 diminished the therapeutic effect of LGALS9 [235]. Key information missing or mismatch due to the tech-
These applications suggest that single-cell multi-omics nology is another issue. Sequencing depth directly affects
technologies are eminently prospective for investigat- the quality of the data obtained, and choosing the appro-
ing neuropsychiatric disorders as well as brain injuries, priate sequencing depth in scRNA-seq is crucial [244].
providing unprecedented opportunities to decipher the Deeper sequencing provides a more comprehensive gene
complexity of the brain. expression profile, while insufficient sequencing depth
In addition to the applications above (Table 3), we may result in the loss of crucial information, impact-
anticipated that single-cell multi-omics and paired bioin- ing the annotation of cell types and the interpretation of
formatics tools would provide a fundamental framework their functions. Besides, some cellular heterogeneities
for the research on a variety of complex diseases or bio- may be not well presented in the transcriptome, and fail
logical processes, including autoimmune diseases [236], to define the state of the cell. High-throughput single-
aging [237], spermatogenesis [238], and others [239, 240]. cell proteomics methods may work, which are not yet
Table 3 Applications in biological research and clinical practice
Technology Diseases Source Main findings Refer-
material ence
Cell atlases construction
scRNA-seq, spatial transcriptomics / Human The study highlighted the transformative potential of single-cell and spatial genomics in understanding disease [181]
mechanisms, and diagnosing, and treating various conditions.
scRNA-seq, scATAC-seq / Human Constructing a human brain organoid development atlas to investigate the regulation of cell fate decisions. [182]
organoids
Wu et al. Biomarker Research

scRNA-seq, scTCR-seq Ovarian cancer (OC) Human A single-cell landscape of the OC ecosystem was depicted and revealed the heterogeneity of functional pheno- [241]
types and developmental origins of macrophages in tumor tissues and ascites.
Developmental biology
Camallia-seq / Mouse A new technology, Camallia-seq, enabled integrative analysis of chromatin accessibility, DNA methylation, tran- [187]
scriptome, as well as cell lineage information, bringing new insights into how cell fate decisions are regulated
and how cell identities are maintained under different modalities.
(2024) 12:110

scRNA-seq, spatial transcriptomics / Human and Precisely characterizing human embryonic limb development over time and space. [194]
mouse
Pathways identification
scRNA-seq, spatial transcriptomics, Cervical squamous cell Human MP6 tumor cells interact with CAFs to shape an immune exclusionary microenvironment via the FABP5-mediat- [195]
spatial proteomics carcinoma (CSCC) ed TGFβ pathway.
scRNA-seq, scATAC-seq Neuroendocrine pros- Human and FOXA2 is a critical driver of the adeno-to-neuroendocrine lineage transition in prostate cancer that can activate [196]
tate cancer (NEPC) mouse the KIT pathway to maintain cancer cell proliferation.
scRNA-seq, snATAC-seq Kidney Disease Human and The Nfix-Ifi27 pathway can cause kidney fibrosis by promoting apoptosis. [198]
mouse
Novel targets discovery
scRNA-seq, snATAC-seq Lymphangioleiomyo- Human and A HOX-PBX gene regulatory network controlled the survival of LAM cells. [201]
matosis (LAM) mouse
scRNA-seq, spatial transcriptomics, Metastatic melanoma Human A TCF4-dependent regulatory network orchestrated multiple transcriptional programs leading to immuno- [202]
spatial proteomics (MM) therapy resistance.
scRNA-seq, spatial transcriptomics Multiple primary lung Human TNFRSF18, associated with non-response to anti-PD-1 therapy was highly expressed in T&NK cells within MPLCs. [203]
cancers (MPLCs)
ICIs
scRNA-seq, scTCR-seq, scBCR-seq Epstein-Barr virus- Human Re-emerged clonotypes in ISG-15+CD8+ T cells after treatment among EBV (+) patients were detected and as- [25]
associated gastric sociated with effector T population expressing CXCL13 in responsive EBV (+) tumor, indicating their significant
cancer (EBV + GC) importance in tumor immunochemotherapy response.
scRNA-seq, TCR-seq Localized bladder tran- Human Identifying distinct CD4+ and CD8+ T cell states within bladder tumors and highlighting the presence of cyto- [206]
sitional cell carcinoma toxic CD4+ T cells and various CD8+ T cell subsets.
(TCC)
scRNA-seq, ACAT-seq Hepatocellular carci- Human and TdLN-TTSM cells were primary memory T cells that respond to ICI treatment, representing the adoptive transfer [207]
noma (HCC) mouse of these cells as a promising immunotherapy approach.
ACT
scRNA-seq, CITE-seq B-cell Acute Lym- Human Lack of TH2 functionality might be the cause of relapse in CAR-T treatment. [209]
phoblastic Leukemia
(B-ALL)
Page 19 of 28
Table 3 (continued)
Technology Diseases Source Main findings Refer-
Wu et al. Biomarker Research

material ence
TCR-seq, CITE-seq Non-small cell lung Human A phenotypic signature based on CD39 and CXCL13 identified neoantigen-reactive T cells in fresh NSCLC. [210]
cancer (NSCLC)
Host-microbe interactions
16 S rRNA sequencing, scRNA-seq Intrahepatic cholan- Human P. fungorum inhibited tumor growth through alanine, aspartate, and glutamate metabolism. [214]
giocarcinoma (ICC)
(2024) 12:110

16 S rRNA sequencing, scRNA-seq Gastric cancer (GC) Mouse Streptococcus anginosus promoted gastric tumorigenesis. [215]
scRNA-seq, TCR-seq Pan-cancer Human and The commensal bacterium Lactobacillus johnsonii enhanced the efficacy of ICI therapy by modulating the stem- [216]
mouse ness of CD8+ T cells.
Infectious diseases
scRNA-seq, scBCR-seq SARS-CoV-2 Human Discovery and characterization of potent neutralizing antibodies from individuals with Omicron breakthrough [145]
infections.
scRNA-seq, BCR-seq, TCR-seq SARS-CoV-2 Mouse A systematical depiction of the immune landscape after vaccinating lipid nanoparticle-mRNA. [146]
scRNA-seq, scCITE-seq, scTCR-seq, SARS-CoV-2 Human Observing unique dynamics in the behavior of specific CD8+ T cells during the recuperation phase from COVID- [219]
plasma proteomics, metabolomics 19, among patients suffering from gastrointestinal sequelae.
Cardiovascular disease
sc-RNA-seq, snRNA-seq, snATAC-seq, / Human Constructing the human cardiac landscape and paving the way for the anatomy and immunology of the heart. [227]
spatial transcriptomics
scRNA-seq, scTCR-seq Myocardial infarction Human and TCR-M cells expressing Treg markers like Foxp3, Il2ra, and Ctla4 suppressed cardiac immune responses post-MI [228]
(MI) mouse and improved cardiac function.
scRNA-seq, CITE-seq Atherosclerosis (AS) Human Deciphering the immune landscapes in the plaques of AS and uncovering immune alterations related to clinical [230]
cardiovascular events.
Neurological and brain disease
scRNA-Seq, proteomics Schizophrenia Human Identifying two disease-associated factors, BRN2 and PTN, which promoted neurogenesis, with PTN also inhib- [233]
organoids ited apoptosis.
scRNA-seq, spatial transcriptomics Ischemic stroke Mouse Two microglial subclusters, ischemic core-associated microglia (ICAM) and ischemic penumbra-associated mi- [24]
croglia (IPAM) were identified in the brains of mice subjected to middle cerebral artery occlusion (MCAO).
scRNA-seq, spatial transcriptomics Ischemic Stroke Mouse LGALS9-CD44 is a crucial pathway after ischemic injury. LGALS9 and CD44 exhibited opposite effects, in which [235]
upregulation of LGALS9 favored recovery from post-ischemic injury, whereas knockdown of CD44 diminished
the therapeutic effect of LGALS9.
Page 20 of 28
Wu et al. Biomarker Research (2024) 12:110 Page 21 of 28

established [131]. Capturing the immune repertoire also Collectively, single-cell multi-omics methods have
faces inherent technical limitations. Instances such as essentially expanded the tools to discover the rich
measuring only TRA or TRB, or erroneous matching to resources and understand the inner workings of biologi-
more than two chains, do occur [25]. Addressing how to cal processes at the single-cell level. Given that future
effectively handle these data requires more uniform rules. multi-omics studies will aid in addressing numerous bio-
Besides, there are various causes for clonal expansion, logical research and clinical practices, the technologies
including the pressure of tumor neoantigens or infec- will become the standard toolkit for studies on molecular
tious diseases. Determining whether clonal expansion is cell biology.
due to a specific factor, like tumor antigen pressure, is a
Abbreviations
topic worth exploring. This may necessitate the creation scRNA-seq Single-cell RNA sequencing
of a dedicated immune repertoire database or the use of scTCR-seq Single-cell T cell receptor sequencing
biochemistry methods to bidirectionally decode TCR and scBCR-seq Single-cell B cell receptor sequencing
scATAC-seq Single-cell assay for transposase-accessible
pMHC, among other approaches. chromatin using sequencing
Doublet refers to two cells encapsulated into one reac- TFs Transcription factors
tion volume [245], which may confound downstream PCA Principal component analysis
UMAP Uniform manifold approximation and projection
analysis, including atlas construction, DEG analysis, and t-SNE t-distributed stochastic neighbor embedding
cell trajectory inference. The emergence of the doublet DEGs Differentially expressed genes
is especially evident when a large amount of cell volume GO Gene ontology
KEGG Kyoto encyclopedia of genes and genomes
is put in. Developing a suitable means of assessing the GSVA Gene set variation analysis
removal of double cells can prevent many strange prob- SCENIC Single-cell regulatory network inference and
lems from arising [245, 246]. clustering
CCA Canonical correlation analysis
Altogether, overcoming these challenges to integrate MMN Mutual nearest neighbors
high-quality, multimodal single-cell multi-omics data is 4sU 4-thiouridine
essential. Understanding the patterns of normal tissue U Uracil
C Cytosine
function and disease progression from a single-cell per- A Adenine
spective is inseparable from these problems being solved. G Guanine
scSLAM-seq Single-cell, thiol-(SH)-linked alkylation of RNA for
metabolic labelling sequencing
Conclusions CMV Cytomegalovirus
Single-cell sequencing provides unprecedented resolu- UMI Unique molecular identifier
tion to identify multicellular connectivity and hetero- EU 5-ethynyl-uridine
EdU 5-Ethynyl-2-deoxyuridine
geneity. Multi-omics technologies have empowered EE Enteroendocrine
the scRNA-seq to break through the original limita- gRNAs Guide RNAs
tions associated with relying solely on transcriptome TME Tumor microenvironment
ISH In situ hybridization
gene expression profiles. Integrated with transcriptome, ISS In situ sequencing
genome, metabolome, proteome, TCR/BCR, epigenome, smFISH Single-molecule fluorescence ISH
etc., and broadening the axes of timescale and spatial seqFISH Sequential FISH
MERFISH Multiplexed error-robust FISH
information, multidimensional information provides RCA Rolling-circle amplification
a comprehensive snapshot of cell types and states. In RCP Rolling-circle product
the present review, we discuss the established cutting- FISSEQ Fluorescent in situ sequencing
STARmap Spatially-resolved transcript amplicon readout
edge single-cell multi-omics technologies over the past mapping
decades. Additionally, burgeoning computational biology 3D Three-dimensional
technologies are another major step toward uncovering CNS Central nervous system
ROI Regions of interest
and deciphering the secrets within the multidimensional LCM Laser capture microdissection
datasets. These bioinformatics tools link the datasets DSP GeoMx digital spatial profiler
from different modalities elucidate the function within Geo-seq Geographical position sequencing
PC Photocleavable
different cell types, and provide a wide range of informa- ICI Immune checkpoint inhibitor
tion through mathematical modeling and artificial intel- NSCLC Non-small cell lung cancer
ligence methods. From the view of clinical practice, we NGS Next-generation sequencing
ST Spatial transcriptomics
highlight the applications in tumor immunology, thera- HDST High-definition spatial transcriptomics
peutic technologies, and drug treatments. Especially for Stereo-seq Spatial enhanced resolution omics-sequencing
the discovery of new cell populations and new targets, as Decoder-seq Dendrimeric DNA coordinate barcoding design
for spatial RNA sequencing
well as evaluation and interpretation of drugs and thera- DBiT-seq Deterministic barcoding in tissue for spatial
peutics, such as PD1 and CAR-T, are elucidated. omics sequencing
Wu et al. Biomarker Research (2024) 12:110 Page 22 of 28

spatial CUT&Tag-RNA-seq Spatial assay of cleavage under targets and AS Atherosclerosis

tagmentation and RNA sequencing AD Alzheimer’s disease
spatial CITE-seq Spatial co-indexing of transcriptomes and
epitopes for multi-omics mapping by highly Acknowledgements
parallel sequencing The schematic diagrams were created by Figdraw (https://www.figdraw.
scWGS Single-cell whole-genome sequencing com/).
SNP Single nucleotide polymorphism
eQTL Expression quantitative trait loci Author contributions
G&T-seq Genome and transcriptome sequencing R.Y. and H.Q.G. acquired the funding. X.Y.W., X.Y., and Y.H.D. wrote this paper
DR-seq gDNA-mRNA sequencing and drew the figures. J.M.Z. and Z.H.Z. made the tables, collected the
5mC 5-methylcytosine references, and reviewed the article. Each author reviewed the final version of
scRRBS Single-cell reduced representation bisulfite the article before giving their approval for publication. X.Y.W. and X.Y. made
sequencing equal contributions to the manuscript.
scWGBS Single-cell whole-genome bisulfite sequencing
scM&T-seq Single-cell methylome and transcriptome Funding
sequencing This work was supported by the National Natural Science Foundation of
scTrio-seq Single-cell triple omics sequencing China (81772710 and 82172691), Jiangsu Provincial Healthcare Commission
ChIP-seq Chromatin immunoprecipitation sequencing Scientific Research Grant Top Project (M2020093), and Jiangsu Provincial Social
scChIP-seq Single-cell ChIP-seq Development Project (BE2023659).
scCUT&Tag Single-cell CUT&Tag
CoTECH Combined assay of transcriptome and enriched Data availability
chromatin binding No datasets were generated or analysed during the current study.
scMS Mass spectrometry-based single-cell proteomics
CITE-seq Cellular indexing of transcriptomes and epitopes
by sequencing Declarations
Treg T regulatory cell
SUGAR-seq SUrface-protein Glycan And RNA-seq Ethics approval and consent to participate
TILs Tumor-infiltrating lymphocytes Not applicable.
LacNAc N-acetyllactosamine
IR Immune repertoire Consent for publication
pMHC Peptide-loaded major histocompatibility Not applicable.
complex
V Variable Competing interests
D Diversity The authors declare no competing interests.
J Joining
C Constant Conflict of interest
CDR3 Complementarity-determining region 3 The authors declare no conflicts of interest.
STARTRAC Single T cell analysis by RNA sequencing and TCR
tracking Received: 29 April 2024 / Accepted: 17 August 2024
LIBRA-seq Linking B-cell Receptor to Antigen specificity
through sequencing
BarBIQ Barcoding bacteria for identification and
quantification
microSPLiT Microbial split-pool ligation transcriptomics References
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