Envisioning the Future of Multiomics

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in Cancer and Immunology
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 Contents
4 41
Introduction Genomic Cytometry and New
Modalities for Deep Single-Cell

5
Interrogation
BY ROBERT SALOMON, LUCIANO MARTELOTTO, FATIMA VALDES-MORA,
DAVID GALLEGO-ORTEGA

Single-Cell Sequencing in

51
Translational Cancer Research and
Challenges to Meet Clinical Diagnostic
Needs Computational Approaches for High-
BY ULRICH PFISTERER, JULIA BRÄUNIG, PER BRATTÅS, MARKUS
HEIDENBLAD, GÖRAN KARLSSON, THOAS FIORETOS Throughput Single-Cell Data Analysis
BY HELENA TODOROV AND YVAN SAEYS

26
Identification of a Tumor–Specific
Gene Regulatory Network in Human
B-cell Lymphoma COVER IMAGE © 10x Genomics

BY 10x GENOMICS

30
Recent advances in single-cell
multimodal analysis to study immune
cells
BY RAYMOND HY LOUIE & FABIO LUCIANI

3
Introduction
F
rom cancer to immunology, single cell RNA-
sequencing (RNA-seq) has dramatically changed how
researchers approach biology. Single cell resolution
has progressed the concept of inherent heterogeneity of
biological systems and led to novel advances in how we
understand developmental processes, treat disease, and
develop therapeutics. Now, biologists can further increase
the breadth of their understanding with multiomic single
cell analysis. In addition to a readout of mRNA abundance
from single cell RNA-seq, single cell techniques can now be
applied to profile DNA, chromatin state, and the proteome.
To take it one step further, some methods enable next
generation multiomics—the ability to capture multiple
measurements simultaneously from the same single cell,
rather than examining one readout at a time. This abundant
data can provide novel insights, but it also presents new
challenges, including how to collect, store, and manage
data; integrate different modalities; and properly interpret
findings.
This collection of articles provides an overview of the exciting
innovations occurring at the forefront of multiomics. The first
two articles focus on oncology. Cancer research is dedicated
to improving cancer diagnostics, patient stratification,
treatment monitoring, and therapeutic development. Single
cell multiomics has provided increasingly detailed cell
atlases that let researchers gain a better picture of tumor
heterogeneity and investigate how that heterogeneity
impacts disease progression and treatment response.
Pfisterer et al. (2020) describes how the latest single cell
multiomic techniques can be applied to cancer research,
reviews the methods available for single cell isolation, and
highlights recent multiomic single cell oncology studies. In
our Data Spotlight from 10x Genomics, the simultaneous
readout of epigenomic and transcriptomic data from the
same cells enables the direct reconstruction of cell type–
specific gene regulatory networks for B-cell lymphoma. This
study highlights the power of using Chromium Single Cell
Multiome ATAC + Gene Expression, the first commercial
solution for paired ATAC-seq and RNA-seq analysis of single
cells. The data from this study is available for download so
you can continue to explore the possibilities yourself.
In Louie and Luciani (2021) our attention shifts to the immune
system, another heterogeneous system that benefits from
single cell investigation. Analysis of multiple modalities,
including chromatin state, transcription status, and protein
4
expression, can provide greater stratification of immune
cell states, including a cell’s ability to bind antigens, attack
invading cells, and follow a path of differentiation. Cell
states can change over time and across space, and multiomic
technologies have been developed to evaluate each of
these variables. This article discusses recent single cell
multiomic applications to immunology, focusing on next
generation multiomic techniques that enable simultaneous
measurement of at least two distinct modalities from the
same single cell. Of particular relevance for immunologists
is the ability to track clonal differentiation of T or B cells
using receptor sequencing in the context of CAR-T therapy,
autoimmune disease, and lineage tracing of hematopoietic
progenitor cells.
The proliferation of multiomic single cell approaches has
been made possible by the confluence of several disparate
technologies, including genomics, microfluidics, cytometry,
and informatics. Genomic Cytometry, described by Salomon
et al. (2020), is any technique that provides cell-by-cell
measurement of multiple modalities, including protein,
mRNA, DNA, and epigenetic states, through a sequencing-
based readout, therefore overcoming the limitations of
fluorescence and mass cytometry by opening up unlimited
analytic space to quantify hundreds of thousands of
different molecular species at once. Multiple methods exist
to perform Genomic Cytometry, including plate-based,
droplet-based microfluidics, solid microfluidics, in situ
combinatorial indexing, image-based approaches, and
spatial transcriptomics.
Gathering data is only the beginning, however. In Todorov and
Saeys (2019), we examine the process underlying analyzing
a single cell experiment, including power calculations
performed during experimental design, inclusion of controls
during data generation, pre-processing, checking for batch
effects during data visualization, cell type identification,
differential analysis, and more. This article reviews methods
for dimensionality reduction and cell clustering, compares
approaches for trajectory analysis, and provides an
introduction to single cell multiomic data integration.
These articles are designed to provide a comprehensive
understanding of the technical innovations happening
right now in single cell multiomics, and highlight how these
advances are fueling the future of biological research and
medicine.
REVIEW ARTICLE

Single-cell sequencing in translational cancer research and

challenges to meet clinical diagnostic needs

Ulrich Pfisterer1,2 | Julia Bräunig1,2 | Per Brattås1,2 | Markus Heidenblad1,2 |

Göran Karlsson3 | Thoas Fioretos1,2,4

1
Center for Translational Genomics, Lund
University, Lund, Sweden Abstract
2
Clinical Genomics Lund, Science for Life The ability to capture alterations in the genome or transcriptome by next-generation
Laboratory, Lund University, Lund, Sweden
sequencing has provided critical insight into molecular changes and programs under-
3
Division of Molecular Hematology, Lund
Stem Cell Center, Lund University, Lund, lying cancer biology. With the rapid technological development in single-cell
Sweden sequencing, it has become possible to study individual cells at the transcriptional,
4
Division of Clinical Genetics, Department of
genetic, epigenetic, and protein level. Using single-cell analysis, an increased resolu-
Laboratory Medicine, Lund University, Lund,
Sweden tion of fundamental processes underlying cancer development is obtained, providing
comprehensive insights otherwise lost by sequencing of entire (bulk) samples, in
Correspondence
Ulrich Pfisterer, Department of Laboratory which molecular signatures of individual cells are averaged across the entire cell pop-
Medicine, Center for Translational Genomics,
ulation. Here, we provide a concise overview on the application of single-cell analysis
Lund University, Lund, Sweden.
Email: ulrich.pfisterer@med.lu.se of different modalities within cancer research by highlighting key articles of their
respective fields. We furthermore examine the potential of existing technologies to
Thoas Fioretos, Division of Clinical Genetics,
Department of Laboratory Medicine, Lund meet clinical diagnostic needs and discuss current challenges associated with this
University, Lund, Sweden.
translation.
Email: thoas.fioretos@med.lu.se

Funding information KEYWORDS

Governmental ALF grants; Lund University cancer research, clinical diagnostics, clinical utility, single-cell sequencing
Cancer Center (LUCC); Medical Faculty Lund
University; SciLifeLab Stockholm;
StemTherapy Lund University

1 | I N T RO DU CT I O N developments in next-generation sequencing.6,7 Clinical applications

Cancer represents highly complex and diverse pathological conditions,
characterized by aberrant genomic, epigenomic and transcriptomic
features, such as structural alterations, single nucleotide and copy
1
number variations (SNVs, CNVs), and altered epigenetic and tran-
scriptional signatures.2,3 Both intra- and intertumoral heterogeneity
contribute to the complexity of cancer with mutations in driver genes
adding on to clonal evolution4 and consequently to dynamic clonal
5
architecture throughout disease progression. Recent years have
witnessed a dramatic progress in studying the genetic and molecular
basis of human cancer, enabled, in part, by the rapid technological
for these platforms span the areas of diagnostics, prognostics and
therapeutics using massively parallel sequencing for whole-genome
(WGS) or targeted DNA-sequencing (eg, whole-exome sequencing
WES), RNA-sequencing, chromatin immunoprecipitation (ChIP)-
sequencing, and DNA methylation assays for epigenetic mapping.
In order to further improve clinical application of sequencing-
based technology and to ultimately provide better cancer diagnosis,
patient stratification, treatment monitoring, and personalized therapy,
the recent initiative of the human tumor atlas network aims at the
generation of longitudinal cell atlases of various tumor types
employing single-cell and spatially resolved technologies.8

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2021 The Authors. Genes, Chromosomes and Cancer published by Wiley Periodicals LLC.

5
 The considerable cellular heterogeneity present in most tumors is
likely to contribute to the currently ineffective and highly individual
responses of patients to therapeutic approaches. While bulk analyses
of tumor tissues have provided important insight into for example, the
transcriptional signature or overall genetic variability of a given
tissue,9-12 it does not resolve the cellular composition of malignant
and normal cells. Hence, resolving tumor composition at single-cell
resolution offers great potential not only to provide critical insights
into tumor biology per se, but also to shed light on other therapeuti-
cally relevant issues related to heterogeneity such as tumor microen-
vironment, cell-of-origin, and cancer stem cells. Thus, the advent of
single-cell analyses promises to improve diagnosis, facilitate monitor-
ing of both disease progression and treatment response and will,
hopefully, pave the way to more personalized therapeutic approaches
to realize the promises of precision medicine (Figure 1A-D).
The importance of elucidating cancer at single-cell resolution has
been demonstrated in a plethora of studies which have allowed inves-
tigators to assess tumor heterogeneity, to define cell types and states
in healthy specimen and tumors, as well as to examine heterogeneous
treatment response and drug resistance, among other clinically rele-
vant applications.13,14
Rapid technological development makes it feasible today to access
15
many different modalities in single-cells, in some cases to profile more
than one measure from a single-cell simultaneously16-23 and to perform
advanced computational analyses.24-26 Several different modalities have
been applied to cancer research using either dissociated single-cells or
intact tissue with spatial resolution (Figure 2). While single-cell sequenc-
ing is progressively applied to study clinical cancer samples, its broader
translation into clinical diagnostics has yet to come.
In order to translate single-cell analyses into reliable clinical appli-
cations, thorough assessment will be essential to define a technology's
overall clinical applicability, which relies on its demonstrated analytical
and clinical validity as well as clinical utility.27-29 Here, we define ana-
lytical validity as the confidence of a given test to measure the pres-
ence or absence of a disease-related alteration. In contrast, clinical
validity is determined as the accuracy and confidence with which a
detected variation can be related to a distinct disease phenotype.
Finally, clinical utility determines whether a test result will yield medi-
cal intervention to ultimately improve the patients' health or, where
treatment is unavailable, support clinical diagnosis of patients.28
This review provides an overview of currently available single-cell
sequencing technologies and how such technologies have been used
recently to provide important insights into the molecular basis of can-
cer. Furthermore, it discusses selected studies of different tumor
types, the results of which suggest that single-cell sequencing will
have great clinical utility in the near future, and highlights challenges
and hurdles that exist in order for single-cell sequencing to meet clini-
cal diagnostic needs.

F I G U R E 1 Schematic representation of different scenarios where single-cell resolution is beneficial. A, Monitoring cellular tumor composition
from diagnosis through treatment to monitor treatment response and to potentially refine therapy. B, Immune profiling of tumors to decompose
the different immune cells and cell states infiltrating the tumor tissue. C, Large-scale analysis of tumor composition to decipher intratumor
heterogeneity as well as heterogeneity of tumors of the same origin among patients. D, Monitoring clonal composition for example during
treatment to determine whether a specific therapeutic approach is efficient

6
F I G U R E 2 Overview of different modalities for single-cell analysis of cancer tissues. Tumor tissues may be analyzed using either tissue-
destructive methods following tissue dissociation or by maintaining the spatial location of the cells in a given tissue. To date, a plethora of
platforms and chemistries exist to access different modalities in single-cells in tumor tissue. They enable metabolome analysis, genome
sequencing, cell surface and immune cell receptor profiling, epigenetic modifications as well as sequencing of the transcriptome. To date, only
certain modalities can be studied at spatial resolution (dotted line), whereas tissue-destructive methodologies are available to study all of the
single-cell modalities depicted

2 | APPLICATION OF SINGLE-CELL mRNA- Combinatorial indexing,50 where individual cells undergo several
SEQUENCING IN CANCER rounds of molecular barcoding, in combination with droplet-based
methods as exemplified in a preprint51 have furthermore greatly
Isolation of single cells may follow different principles: individual cells increased the number of cells which can be profiled in a single experi-
may be handpicked or sorted into PCR plates by flow cytometry. It is ment. This elevated throughput leveraged large-scale studies as exem-
further possible to directly dispense cells into chips harboring several plified by profiling 690 000 single-cells of the adult mouse brain
thousand nanowells, to trap single cells in channels and capture sites giving rise to a comprehensive cell atlas of the rodent brain.52 While
of microfluidic devices, as well as to encapsulate single cells in nano the most relevant single-cell genomics applications have been used in
oil-droplets using yet other microfluidic devices. With a growing a plethora of different studies53 and even have been subjected to sys-
demand to study large cell numbers, microfluidic devices for droplet- tematic comparison regarding cost and information content,54,55
based cell capturing are at present among the most common single-cell transcriptomics is still in the early stage of clinical transla-
platforms used. Importantly, the principle of cell isolation does not tion and application.
necessarily restrict the modalities which can be analyzed. An overview One of the very first attempts to assess the transcriptome of single
of the most widely adopted single-cell isolation principles, platforms cancer cells was described with the development of the original Smart-
and modalities can be found in Table 1. seq chemistry.56 The potential of single-cell RNA-sequencing for diag-
Single-cell transcriptomics has greatly increased our understanding nostic purposes was initially demonstrated on a metastatic breast can-
of the composition of complex tissues30-33 and has facilitated the study cer cell line (MDA-MB-231) by monitoring clonal evolution manifested
of a wide variety of human diseases at unprecedented depth.34-39 At in transcriptional alterations and mutation analyses inferred from
present, a large number of single-cell transcriptomics methodologies mRNA reads along treatment with Paclitaxel, which provided novel
40 41 42
and platforms are at hand (Smart-seq2, Smart-seq3, STRTseq, insight into drug resistance dynamics.57 Furthermore, single-cell
43 44 45 46
Cyto-seq, inDrop, Drop-seq, 10X Genomics as well as CEL- sequencing of lung adenocarcinoma (ADC) cells identified a distinct
seq2,47 Quartz-seq,48 MARS-seq,23 Seq-Well49). transcriptional signature of cells associated with resistance to anti-

7
TABLE 1 Overview of different single-cell isolation principles and corresponding platforms most commonly used in the cited literature of this
review

Modalities studied in selected references

Single-cell isolation principle Examples of platforms or chemistries applying various platforms
Manual isolation and dispensation into Serial dilution SNV
tubes or plates Hand picking mRNA, inferred SNV
Mouth pipetting mRNA, TCR expression, SNV, CNV,
methylome
Fluorescence-activated cell sorting into Various chemistries (eg, Smart-seq2) mRNA, TCR expression, SNV, CNV,
tubes or plates methylome, ATAC
MARS-seq mRNA
TCR-seq TCR expression
QRP DOP-PCR CNV
Immunomagnetic cell separation MagSweeper SNV, CNV
Cell dispensation into nanowells iCell8cx mRNA, DNA
cellenONE, CNV
sciFLEXARRAYER S3
Seq-well mRNA
Microfluidics with capture sites Fluidigm C1 mRNA, SNV, CNV, ATAC
DEPArray (Menarini Silicon Biosystems) CNA
Microfluidics with nanodroplets 10X Genomics mRNA, TCR expression, inferred SNV/CNV,
ATAC, cell surface proteins
inDrop mRNA, TCR expression
MissionBio SNV/CNV, cell surface proteins
Custom-built Chromatin immunoprecipitation

Abbreviations: ATAC, assay for transposase-accessible chromatin; CNV, copy number variation; MARS-seq, massively parallel RNA single-cell sequencing;
QRP DOP-PCR, quasi-random priming degenerate oligonucleotide primed polymerase chain reaction; SNV, single-nucleotide variant; TCR, T cell receptor;
TCR-seq, T cell receptor sequencing.

cancer drugs.58 Similarly, single-cell resolution of the transcriptome in
renal cell carcinoma has shed new light on intratumor heterogeneity
and led to the derivation of a new, combinatorial therapeutic strategy.
Moreover, two single-cell studies assessed the transcriptome of circu-
60
lating tumor cells (CTCs) in prostate cancer (PC) and breast cancer.
This led to the identification of two distinct phenotypes of breast can-
cer CTCs with the capacity to interconvert and potentially contribute
to treatment resistance.61 Single-cell analysis further revealed great
diversity of PC CTCs among treated patients and identified splice vari-
ants and mutations in the androgen receptor (AR) gene, associating
failed AR inhibitor treatment to noncanonical Wnt signaling.60 Overall,
analysis of single CTCs may open up for exciting possibilities for future
non-invasive, single-cell diagnostics.
Tumor heterogeneity has been elucidated in glioblastomas,
breast cancer,62 and large-scale tumor cell atlases have been gener-
ated in lung,63,64 renal,65 and pediatric brain tumors66 at single-cell
resolution. Interestingly, validating single-cell transcriptomics data
with bulk RNA-sequencing, proteomics and functional studies con-
firmed novel phenotypes of endothelial cells which in turn potentially
opens up for new therapeutic target points blocking tumor angiogene-
64
sis in lung cancer. Further technological advancement leveraging
increased cellular throughput, led to the identification of discrete tran-
scriptional programs and cellular compositions in relation to increasing
clinical grade tumors of glioma,37 distinct transcriptional programs of
tumor-associated macrophages in glioma67 and to the determination
59
of varying gene signatures in malignant cells in head and neck squa-
mous cell carcinoma.68 Unbiased clustering of single-cells obtained
61
from colorectal cancer not only discovered novel cancer-associated
fibroblast types and unmasked tumor heterogeneity, but importantly
also demonstrated that single-cell transcriptomics provides prognostic
insight previously hidden in bulk sequencing data.69 Furthermore, dec-
iphering of cellular composition of breast cancer patient-derived
xenografts (PDX) identified a stem-like cell type with high epidermal
growth factor receptor (EGFR) gene expression levels and further
linked high EGFR expression to an elevated mesenchymal gene
signature,70 similar to another study identifying elevated expression
38
of epithelial-to-mesenchymal (EMT) - associated genes in breast can-
cer cells.71 Following breast cancer samples along the treatment
course of several years, integrated single-cell genome and trans-
criptome analyses identified discrete phenotypes associated with
chemoresistance, with the most prominent upregulation being an
EMT gene signature.72
While combined analysis of DNA and RNA in individual cells is
feasible,22 current protocols are not amendable for high cellular
throughput and have therefore not frequently been used. Comple-
mentation of gene expression with inferred CNV from full-length

8
single-cell mRNA to distinguish malignant cells37,38,71,73,74 or targeted
genotyping35 present attractive alternatives to comprehensively study
human malignancies. Accordingly, chromosomal aberrations charac-
38
teristic for glioblastoma were inferred onto tumor cells and classifi-
cation of malignant cells in glioma were corroborated.37 In line with
this, cancer-specific genomic aberrations could be inferred from
single-cell transcriptomics data and were restricted to malignant gli-
oma cells. Haplotype inference additionally revealed heterozygous
loss of chromosome 14 alleles in glioma tumors.74 Interestingly, RNA-
inferred CNV information clearly distinguished immune from carci-
71
noma cells in breast cancer, opening up for the possibility for unre-
strained profiling of both cell types without usage of cell surface
markers. Deducting genomic alterations such as CNV from mRNA-
sequencing also aided the delineation of cellular hierarchies in
75
oligodendroglioma.
More recently, high throughput single-cell mRNA approaches
such as Seq-well49 combined with targeted genotyping were used to
elucidate molecular hierarchies in acute myeloid leukemia (AML) and
confidently identified six malignant AML cell types with mutations
being absent in healthy donor samples.76 This study furthermore
combined both short- and long-read sequencing technologies to
determine genetic aberrations such as insertions, deletions and gene-
fusions in individual cells. It additionally employed a large cohort of
longitudinally collected AML samples (diagnosis, treatment,
remission),76 thereby suggesting that single-cell transcriptome analy-
sis may be applied to monitor treatment response and putatively aid
clinical decision making. However, in order for this approach to pro-
vide analytical and ultimately clinical validity, it needs to possess
greater detection sensitivity of mutation signatures. Hence, while this
work leveraged large-scale analysis, about 40% of the targeted sites
were not detected and mutations located in proximity to either the
30 end of the mRNA or to an internal polyadenylation site were cap-
tured more efficiently. This is directly linked to the design of the
sequencing library preparation in Seq-well49 which preferentially
yields sequences toward the 3 end of mRNA transcripts via polyT-
0
capture sequences. In line with this, Petti and co-workers utilized a
droplet-based platform to infer genomic information from single-cell
transcriptomes and were able to deduce SNV information in 23% of
77
the cells analyzed. In addition, the authors confidently distinguished
normal from tumor cells and successfully identified a cell-surface
marker (CD99) from the single-cell transcriptomics data, enabling for
the precise isolation of distinct clonal cells.77 While this study fell
short in identifying novel cell-surface markers, it nicely demonstrated
the possibility for precise isolation of malignant cells for refined
downstream analyses. Recently, uveal melanoma (UM) was studied
by integrated mRNA and B and T cell receptor (BCR and TCR)
78
expression. Inferring genomic aberrations present in the single-cell
transcriptomics data using the software inferCNV, both canonical
and non-canonical CNVs were identified across all samples, delineat-
ing clonal structures in the tumor tissue.78 This furthermore demon-
strates the applicability of single-cell transcriptome analysis to
deduce genomic variation in cancer.
Single-cell transcriptomics is a rapidly evolving technology which
already has yielded critical insight into cellular diversity of complex tis-
sues.31 The highlighted research in this section provided first insights
into pathological transcriptional changes underlying cancer develop-
ment and progression as well as response to therapy. These studies
clearly demonstrate a great potential for single-cell mRNA-sequencing
to become a clinical diagnostic tool in the near future. Most likely, the
first clinical applicability will be as a prognostic tool in the diagnostic
setting, for example in hematologic malignancies, to decipher the cel-
lular composition of normal and malignant tissue based on their tran-
scriptional signatures. However, this will require several large-scale
studies to demonstrate that cellular composition correlates with
important clinical parameters. Along with increased sensitivity and
reproducibility, single-cell mRNA-sequencing, in combination with
other modalities (see below) is likely to become increasingly important
in monitoring treatment response.
3 | SINGLE-CELL IMMUNE PROFILING IN
CANCER
In the thymus, lymphoid progenitors are molded into committed T
cells which in turn play an important role in shaping the adaptive
immune system. Besides the acquisition of somatic mutations
throughout life in normal cells of different tissues, contributing to
cancerogenesis, progressive decline in T cell production in the thymus
has been associated with an increased incidence of age-relate dis-
eases, including cancer.79 Moreover, the type of immune cells and
their location and density in a given tumor were postulated to possess
prognostic value, and suggested that high frequency of cytotoxic
memory T cells in a tumor tissue was indicative of disease relapse post
treatment.80 These results exemplified the potential clinical benefit of
comprehensive immune cell profiling of tumors and strengthened the
ultimate necessity to retain spatial information within the tumor
tissue.
Since the development of T cells involves both the differentiation
of T lymphocytes and the generation and maintenance of a diverse
TCR repertoire, precise comprehension of developmental processes
underlying T cell specification are of significant importance to under-
stand disease progression in cancer. While targeted TCR analysis via
nested PCR has allowed analysis of several hundreds of single-cells,81
recent developments have made it possible to probe even larger num-
bers of T cells in an unbiased fashion,73,82,83 as well as in combination
with targeted TCR analysis.84 In addition, simultaneous profiling of
both transcriptomic and TCR signatures from thousands of individual
cells has been reported.85-87
A recent study revealed bias in VDJ gene usage during recombi-
nation of TCRβ throughout differentiation toward mature T cells by
integrating transcriptional signatures of cell states with the expression
data on TCR chains α and β.88 This observed bias in TCR recombina-
tion might impact the adaptive immune response and consequently an
individual's response to antigenic stimuli.
9
 In the attempt to elucidate the tumor microenvironment, single-
cell analysis enabled the identification of molecular signatures of
exhaustion programs in T cells, their associated markers, and linked
dysfunctional signatures to tumor reactivity in human mela-
noma.73,84,85 It further led to the determination of a transcriptional sig-
nature of specific immune cells which could in turn be linked to patient
survival and improved existing prognostication of breast cancer
patients.89 Moreover, integrated mRNA- and targeted TCR-sequencing
revealed that dysfunctional T cells exhibited prominent clonal expan-
sion with continuous proliferation in metastatic melanoma.84
Single-cell analysis further defined clonotypes of T cells while
suggesting their activation status in the human hepatocarcinoma
(HCC) microenvironment,90 and identified a distinct dendritic cell type
capable of migrating from the tumor tissue to the hepatic lymph
node.91 Furthermore, valuable insight into transcriptional signatures
of tumor-infiltrating myeloid cells in lung ADC has been obtained
using high throughput single-cell mRNA-sequencing.92
A recent study utilized single-cell technology to elucidate the
composition of immune cells in the tumor microenvironment of breast
86
cancer. High-throughput integrated mRNA- and TCR-sequencing rev-
ealed an increased phenotypic diversity of both lymphoid and myeloid
cells in tumorous tissue, as opposed to normal breast tissue, and
86
exhibited inter-patient variation in metabolic signatures. Corroborating
their findings using two different platforms (inDrop and 10X Genomics),
the authors identified continuous T cell activation, which in part could
be explained by broad stimuli activating TCR repertoire, and showed
that tumor residing T cells were comprised of different clonotype clus-
ters with varying activation states.86 Overall, distinct phenotypes are
shaped by the TCR repertoire in response to antigenic stimuli but diver-
sity is also mediated by environmental stimuli such as hypoxia.86
More recently, integrated analysis of mRNA and TCR repertoires
in 141 623 T cells was performed in four different types of cancers
(non-small-cell lung ADC, endometrial ADC, colorectal ADC and renal
clear cell carcinoma) as well as in histologically normal adjacent tissue
(NAT) and peripheral blood.87 This study led to the discovery that
diverse clonal expansion patterns across patients with clonotypes being
either expanded similarly in the tumor and NAT or following differing
patterns.87 Moreover, this work shed light on the existence of a strong
correlation between peripheral and intratumoral clone size, a finding
which was substantiated by re-analyzing data of related studies investi-
gating T cells in non-small-lung cancer93 and colorectal cancer cells.94
In addition, non-exhausted T cell clones were more likely to be blood-
associated as opposed to exhausted clones and different clonal expan-
sion patterns were correlated with the clinical response of patients.87
These findings suggest that the detection of clones in blood may
serve a useful proxy to determine the presence of clinically relevant,
expanded clones in the tumor, opening up for the possibility of “liquid
biopsies” for monitoring treatment response following therapy with
Atezolizumab, Sunitinib, or IMmotion150 using single-cell technology.
Utilizing the same combined mRNA- and TCR-sequencing
approach on basal and squamous cell carcinoma samples before and
after immune checkpoint blockade (ICB) treatment suggested that
novel T cells exert treatment response rather than T cell clones
10
pre-existing in the tumor.95 Analysis of patients with metastatic mela-
noma responsive to ICB treatment displayed a greater fraction of
large T cell clones as opposed to non-responsive patients.85 Interest-
ingly, transcriptional alterations and gene modules induced by ICB
treatment did not correlate with the clinical outcome observed in
patients,85 rendering simultaneous profiling of TCR clonality a neces-
sity to deduct clinically relevant information.
Despite the correlation of therapy response to clone size, T cell
clonal specificity to distinct tumor antigens yet needs to be deter-
mined and integrated to define lasting predictive markers for the out-
come of different ICB therapies. Interestingly, TCR repertoire
analysis of CD8+ T cells in UM revealed that these cells strongly
expressed the checkpoint marker gene LAG3, whereas, unexpectedly,
expression of PD1 was minimal.78 This may in part explain the lack
of responsiveness of UM to checkpoint immunotherapy targeting
PD1. Moreover, single-cell analysis of immune cells from glioblas-
toma combined with murine models identified a distinct macrophage
type which in turn appeared to be a potential target for combinato-
rial immune therapy.96
Very recently, the development of single-cell metabolic regulome
profiling (scMEP) made it possible to study the highly dynamic func-
tions exerted by immune cells manifested in metabolomic alterations
at spatial resolution, deciphering metabolic profiles of CD8+ T cells in
the tumor microenvironment.97 The ability to analyze immune cell
migration into diseased tissue, which is tightly regulated by the cells'
metabolism, holds great promise to understand immune cell-mediated
processes in the tumor following treatment. In addition to study
tumor immune cells based on mRNA and TCR expression or metabo-
lites, recent technological advances made it possible to generate cell
atlases of human tumors based on the expression of cell surface
markers complemented with single-cell transcriptome sequencing,
exemplified by the analysis of lung ADC.98
Taken together, single-cell immune profiling holds great potential
to refine existing therapies (Figure 1A) and has greatly increased our
understanding how clonal composition of immune cells, both within
the tumor and adjacent tissue, is encoded in the transcriptome and
receptor repertoire (Figure 1B). Single-cell resolution has further
offered insight into intra-tumoral heterogeneity of immune cells and
potential bias in responsiveness to treatment, how regulation of meta-
bolic pathways underlies immune cell function, and how these path-
ways may be exploited to device novel therapeutic strategies
enhancing the overall immune cell response. Given the dramatic
impact of ICB in cancer treatment during recent years and the realiza-
tion that the immune system plays a critical role in cancer develop-
ment and progression, single-cell immune profiling is most likely to
become one of the first strategies reaching clinical diagnostics.
4 | EP I G E N E T I C A NA L Y S E S O F C A N C E R A T
S I N G L E - C E L L RE S O LU T I O N
Besides immune infiltration, transcriptomic and genomic alterations,
epigenetic changes underlie cancer development and evolution, but
also disease prognosis and treatment outcome.99,100 Epigenetics con-
stitute inheritable cellular regulation of gene expression, which occur
independently of the genetic information. Chromatin status, accessi-
bility and conformation are highly regulated by histone and genome
modifications, and by interactions between DNA and protein struc-
tures. DNA methylation and histone acetylation have been the subject
to intensive research. Hypermethylation of promoter regions, a gen-
eral reduction in genomic 5-methylcytosine levels as well as the loss
101,102
of histone acetylation are commonly observed in cancer cells
and ultimately contribute to altered gene expression regulation. In
contrast to genomic mutations and aberrations, epigenetic marks and
their deregulation are often reversible.
To date, several DNA methyltransferase inhibitors (DMTIs)
and histone deacetylase inhibitors have been investigated as anti-
103
cancer drugs and are approved by the FDA for several cancers.
First trials with DMTIs yielded promising treatment results, but they
also evoked severe side effects.104,105 Lower treatment dosages
of DMTIs were similarly successful, but no major demethylation
effect was observed in bulk sequencing experiments in contrast
to higher treatment concentrations.106,107 Analysis of monoclonal
populations of the human colon carcinoma cell line HCT116 showed
that every clone has a distinct partial demethylation pattern and
that the resulting changes in epigenetic regulation are sufficient to
slow cancer cell proliferation.107 This monoclonal analysis exempli-
fied the necessity for single-cell resolution in cancer epigenetics
in order to unravel cellular heterogeneity, to device novel therapies
and to monitor treatment. Today, several single-cell methods for
DNA methylation and chromatin accessibility are available to
study cancer (scATAC-seq, sciATAC-seq, scRRBS-seq, scChip-seq,
scTrio-seq).108-112
Single-cell reduced-representation bisulfite sequencing (scRRBS-
seq) was used to trace cancer evolution by measuring alterations in
the methylome in both healthy individuals and patients with chronic
lymphocytic leukemia (CLL) before and after treatment.113 Overall,
this study revealed impaired B cell development in diseased individ-
uals and increased cell-to-cell heterogeneity of B cells in CLL as
opposed to healthy controls and normal B cells.113,114
The application of single-cell assay for transposase-accessible
chromatin sequencing (scATAC-seq) showed that breast cancer cell
lines clustered separately before and after JQ1-treatment based on
their epigenetic state.115 Furthermore, scATAC-seq identified a sub-
population of a PD-1 immunotherapy responsive T cell population
116
and its underlying regulatory mechanism in basal cell carcinoma,
and has pinpointed distinct transcription factor motifs which drive
117
cancer heterogeneity in leukemic cells.
Unlike scATAC-seq, single-cell chromatin immunoprecipitation
(scChip-seq) also captures repressed regions of the chromatin in addi-
tion to accessible sites.110 Using this approach, a recent study con-
cluded that tumor cells resistant to the cytostatic drug Capecitabine
can be discriminated from non-resistant tumor cells based on their
chromatin status in a triple-negative breast cancer model, and that
distinct repressed H3K27me3 regions were associated with genes
responsible for therapy resistance.118
Besides monomodal single-cell approaches, multimodal methods
offer the possibility to assign an epigenome to a transcriptome,
genome, or proteome revealing the regulatory correlations between
them. Several scATAC-seq and single-cell bisulfite sequencing proto-
cols provided enough genome coverage to analyze CNVs.112,116 One
of the earliest single-cell study in cancer epigenetics utilized bisulfite
sequencing, CNV and transcriptome analysis (scTrio-seq) to investi-
gate hepatocellular carcinoma (HCC).112 The authors found that the
event of a CNV did not alter the methylation pattern of the affected
DNA region and that aberrantly methylated regions did not overlap
with the presence of CNVs, but that both influenced transcriptional
levels. These results additionally confirmed that DNA methylation in
promoter regions correlates negatively with gene expression, whereas
DNA methylation in the gene body correlates positively with tran-
scription as demonstrated in HepG2 and HCC cells. However, only
26 single HCC cells from one patient were investigated, thus limiting
the general conclusions possible to be drawn for HCC from this
study.112
ScRRBS-seq and Smart-seq2 data were obtained from the same
cell by separating mRNA and DNA, revealing an Ibrutinib sensitive B
cell subpopulation in CLL patients, which is expelled from the lymph
node upon treatment.113
Combining CITE-seq, Smart-seq2, and scATAC-seq to investi-
gate mixed-phenotype acute leukemia (MPAL) revealed that ana-
lyses based on either surface-protein expression, chromatin
accessibility or mRNA expression yielded reproducible and compa-
rable cell clusters.108 While MPAL is a rare disease displaying char-
acteristics of both AML and acute lymphoblastic leukemia (ALL),
MPAL patients are more responsive to ALL treatment compared to
AML therapies.119 Single-cell ATAC-seq and Smart-seq2 data pro-
vided the necessary resolution to show that distinct genes are uni-
versally upregulated in either MPAL or AML cancer cells,108
possibly explaining why AML treatments often fail in MPAL
patients. In addition, RUNX1 was associated with transcription fac-
tor binding motifs in MPAL cancer cells.108 Using single-cell combi-
natorial indexing ATAC-seq (sciATAC-seq), the potential regulatory
role of RUNX transcription factor motifs was investigated in a
murine lung ADC model, revealing that accessible RUNX transcrip-
tion factor motifs were mainly present during the metastatic stage.
Additionally, transcription factor scores were matched with differ-
ent tumor stages, as well as RUNX and NKX2.1 transcription factors,
which correlated with patient prognosis.109 Interestingly, while the
transcription factor NKX2.1 is used as a diagnostic marker in clinical
lung ADC,120 the metastatic sciATAC-seq cluster correlated better
with overall patient survival than the NKX2.1 cluster,109 suggesting
that the accessible chromatin status could be used as an improved
diagnostic marker.
Besides genomic DNA, mitochondrial DNA (mtDNA) is subjected
to epigenetic alterations, SNVs and CNVs, which play a role in tumori-
genesis, cancer progression and drug resistance.121 Modification of a
droplet-based scATAC-seq protocol facilitated capturing of mtDNA
(scmtATAC-seq) and demonstrated that a 50x coverage of the mito-
chondria genome can yield robust CNV and even SNV data in addition
11
to accessible chromatin information.122 This revealed mutations and
CNVs related to disease progression and drug resistance in CLL
patients, with individual subpopulations showing impaired methyla-
tion patterns in genes related to drug resistance such as TIAM1 and
ZNF257. Interestingly, the small size of the mitochondrial genome
with only 16 kb in size strongly reduces sequencing costs, potentially
facilitating broader application areas.
At present, published single-cell studies in the field of cancer epi-
genetics have demonstrated that available methods and protocols are
sufficient to distinguish between healthy and diseased cell types, and
to enlighten cancer heterogeneity, progression, and treatment effects.
Identified subpopulations, transcription factor motifs, and regulatory
mechanism could potentially predict patient outcome and drug resis-
tance suggesting sufficient analytical and potentially even clinical
validity. However, as this is a relatively young field, modalities need
further refinement to accomplish analytical validity. ScRRBS-seq
covers less CG islands than bulk bisulfite sequencing110 and in com-
parison with single-cell bisulfite sequencing methods, scChip-seq has
an overall lower genome coverage and a higher ratio of background
noise.123
Some methods, like scTrio-seq, offer a lower throughput
impeding the possibility to access cancer heterogeneity in its
entirety. In the recently developed method Cleavage Under Targets
and Tagmentation (Cut&Tag), antibodies target defined histone
modifications and conjugated Tn5 cuts accessible DNA, which
reduces unspecific signals. Overall, analytical validity of novel
124
methods such as Cut&Tag remains to be demonstrated in cancer
research.
Finally, integration of epigenetic modifications with other modali-
ties such as mRNA or cell surface protein expression will be of impor-
tance to gain more complete insight on how the disease is manifested
and regulated, as well as to explain the effect of cancer-induced epi-
genetic changes.
5 | A SS ES SM E N T OF C LO N A L
H E T E R O G E N E I T Y I N CA N C E R B Y
S I N G L E - C E LL D N A - S E Q U E N C I N G
Continuous gain of genetic variation in individual cells underlie tumor
initiation, maintenance and evolution. In particular, ongoing cell divi-
sion within tumor tissue fosters genetic mosaicism manifested in
1
CNVs, SNVs and gene breakpoints. While bulk DNA-sequencing has
demonstrated substantial genetic heterogeneity in cancers, such as
125 126
AML or primary renal carcinomas, determination of clonal struc-
ture of cancer types necessitates single-cell resolution.
Among the first methods to be used to interrogate clonal diversity
at single-cell resolution were PCR-based methods such as degenerate
127
oligonucleotide primed PCR (DOP-PCR), isothermal multiple dis-
placement amplification (MDA)128-130 as well as PicoPlex131 and mul-
132
tiple annealing and looping-based amplification cycles. Increased
130
cellular throughput was achieved by employing microfluidic devices
and single-cell combinatorial indexed sequencing (sci-seq),133 with
12
microfluidics enabling stringent quality control via cell imaging, while
simultaneously reducing contaminating ambient DNA interfering with
genomic analyses.
Further optimization in part addressed the shortcomings of exis-
ting approaches with regard to low genomic coverage and allelic drop-
out rates, lack of uniformity, and polymerase-induced errors.134 As
such, recent methods have utilized DNA transposition in combination
with linear amplification135 or direct construction of sequencing-ready
libraries.136,137
Existing technologies facilitate the investigation of CNVs and
SNVs, however, other structural variations such as translocations and
inversions - relevant measures of disease prognosis - are more chal-
lenging to identify at single-cell resolution. Strand-seq enables the
generation of directional sequencing libraries and strand-specific
sequencing reads, yielding homolog resolution in single cells.138 This
was recently utilized to investigate evolutionary differences between
human and macaque based on genetic inversions139 and to develop
the analytical tool single-cell tri-channel processing (scTRIP)140
extracting and utilizing additional information from Strand-seq data.
While this approach enables for more comprehensive analysis of
genomic complexity, it relies on the possibility to label nascent DNA
during replication, which excludes its application to clinical samples
containing non-dividing cells or nuclei.
Single-cell DNA-sequencing has been used intensively to deci-
pher clonal structures in different cancer types and to augment our
knowledge on tumor clonal evolution. An early study applied DOP-
PCR on 100 single nuclei isolated from two human breast cancer
cases and demonstrated that clonal evolution patterns can be inferred
from shallow single-cell WGS.127 While this study did not provide suf-
ficient coverage to resolve SNVs in a genome-wide manner, subse-
quent utilization of G2/M nuclei yielded comparably higher genome
coverage and improved both allelic dropout and false positive rate in
breast cancer samples.141 In this study, the authors indicated that
structural genomic alterations occur early during breast cancer evolu-
tion, while SNVs are acquired progressively and gradually contribute
to clonal diversity.141 The finding, that the majority of single-cell
CNVs were clonal and stable during tumor growth of breast
cancer,142 additionally strengthened the notion that copy number
aneuploidy is acquired early during tumor evolution. Single-cell analy-
sis of breast cancer xenografts moreover corroborated that clonal
expansion dynamics represent reproducible trajectories, indicating
that clonal selection follows a non-random process with distinct muta-
tion genotypes defining clonal fitness and therefore clonal expansion
processes.143
In longitudinal breast cancer samples, bulk exome sequencing
integrated with single-cell DNA and RNA analyses provided insight
into clonal extinction in response to treatment and identified resistant
clones selectively expanded as a result to chemotherapy.144 Single-
cell analysis furthermore enabled the identification of patient-
individual clonal seeding patterns in colorectal cancer leading to the
metastatic tumor.145
Highly relevant with regard to clinical application was the dis-
covery that a large fraction of both trunk and metastatic mutations
could be recapitulated in CTCs from PC146 and that CNV pattern
on a whole-genome scale of CTCs was not altered during the treat-
147
ment course of lung cancer. Furthermore, CNVs detected in
CTCs of ADC and small-cell lung cancer (SCLC) were reproducible
between cells and individuals.147 In line with this, copy number
aberrations in CTCs of SCLC were used to determine classifiers
supporting categorization of chemosensitive or chemorefractory
148
SCLCs. Single-cell WGS of 88 CTCs generated classifiers with
sufficient power to assign the vast majority (>80%) of CTC test
samples to either a chemosensitive or chemorefractory treatment
148
response. This suggests an exciting possibility for single-cell
analysis to provide analytical validity for future diagnostic purposes
similar to single-cell transcriptome studies targeting CTCs,60,61
especially in the absence of primary tumor tissue. However, in
order to reach closer to clinical validity and utility, the persistence
of CNV patterns in lung cancer CTCs needs to be corroborated in
larger patient cohorts. In addition, molecular classifiers capable of
predicting treatment response of SCLCs will require a larger
starting number of cells covering a more complete space of geno-
mic alterations.
Single-cell sequencing of hematologic malignancies such as AML
gained insight into the clonal architecture underlying this heteroge-
nous disease entity, although in limited sample numbers.131 In con-
trast, droplet-based cell capturing and barcoding opened up for the
possibility to profile known genomic loci in AML at unprecedented
throughput.149,150 More recently, a similar approach leveraged analy-
sis of 735 483 single-cells obtained from 123 AML patients, unveiling
clonal evolution patterns and correlation of AML driver mutations.151
In total, a selection of 530 validated mutations were included in the
analysis, which in the case of a subset of longitudinal AML samples,
provided additional insight into the clonal evolution processes during
treatment.151 Furthermore, a very recent study utilized droplet-based,
targeted single-cell DNA-sequencing in AML on a large cohort of sam-
ples, providing insight into clonal complexity and co-occurring muta-
tions in epigenetic modifiers in AML along with changes in cell surface
protein expression underlying the pathogenesis of clonal
152
hematopoiesis.
Integration of bulk exome and whole-genome sequencing on
51 cases of childhood ALL identified aberrant RAG recombinase activ-
ity as critical driving force for genomic aberrations underlying leuke-
153
mic transformation. Targeted genotyping of mutations and
structural variants derived from bulk exome sequencing allowed the
construction of phylogenetic trees. This confirmed that the fusion
gene ETV6-RUNX1, which is considered one of the initiating genomic
153
lesions in this form of ALL, was found in the root of both trees.
Indication for RAG-mediated deletions in cells spanning the entire
phylogenetic tree further suggested that the genomic aberrations
observed were formed through a continuous process in these two
153
cases. Shortcomings of this study comprised the limited number of
single-cells processed; a relatively small number of genomic lesions
were analyzed and the dropout rates of mutant alleles were not thor-
oughly assessed. In contrast, microfluidic MDA targeted genome
sequencing of six patients of ALL provided higher cellular throughput
(1479 single-cells) and combined different computational approaches
for the identification of clonal structures and the removal of low qual-
ity cells due to WGA-induced noise.154 This allowed the authors to
identify clones co-occurring in most patients and to suggest a more
precise hierarchical clonal structure for ALL where the majority of
structural aberrations preceded point mutation acquisition and VDJ
recombination.154
The literature summarized above clearly demonstrates that
single-cell DNA-sequencing is capable to provide analytical validity,
for example, in elucidating tumor heterogeneity, and to monitor
clonal evolution in response to treatment (Figure 1A,C,D), features
of importance in personalized medicine. Particularly, in cases with a
high prevalence of genomic lesions specific for a given cancer type,
targeted genomic approaches hold great potential to become rou-
tine diagnostic application in the near future. Finally, it will be of
essence to understand how different clones and their respective
expansion patterns influence tumor evolution and treatment
response.
6 | SP A TI A L R E SOL U T I ON TO A I D C A N CE R
T I S S U E A NA L Y S E S A N D D I A G N O S T I C S
In order to truly understand tumor behavior, particularly in solid can-
cers, both disease-related transcriptional and genomic alterations
need to be related to the cells' phenotypes in the spatial context of
the tumor microenvironment. Retaining information of type, density
and location of immune cells in colorectal cancer tissue demonstrated
an association of spatial immune cell composition with clinical out-
come.80 It was suggested that such immunological criteria could be
relevant for a clinical application in cancers where the density of
tumor-infiltrating T cells is linked to favorable prognosis. Current tech-
nologies for massively parallel processing of mRNA or genomic alter-
ations lack spatial resolution as a consequence of tissue dissociation,
which in addition has been shown to potentially induce misleading
transcriptional signatures.155
Different approaches have evolved to profile mRNA or protein
expression with spatial resolution while either preserving the tissue
structure156-159 or destructing it by usage of molecular tags providing
spatial information,160 imaging mass cytometry (IMC),161 or laser cata-
pulting.162,163 Co-detection by indexing (CODEX) allows for highly
multiplexed profiling of protein markers and has been used to deci-
pher differences in tissue composition in murine normal and diseased
spleen at single-cell resolution.159 Its applicability to clinical human
samples, however, still needs to be shown in large-scale studies.
Moreover, multiplex immunohistochemistry has enabled parallel visu-
alization of distinct immune checkpoint molecules at single-cell
resolution.164
The GeoMx/DSP platform has been applied to identify protein
markers associated with treatment outcome in melanoma,156 to evalu-
ate the PC micro-environment,158 and to assess B and T cell pheno-
types in melanoma tumors.165 Furthermore, this platform has been
used to study B cell localization in tertiary lymphoid structures using
13
T A B L E 2 Overview of technical details of translational research articles highlighted in this review which describe transcriptome or immune
profiling of single-cells

14
Abbreviations: ADC, lung adenocarcinoma; AML, acute myeloid leukemia; ATRT, atypical teratoid/rhabdoid tumors; BCC, basal cell carcinoma; BCR, B cell
receptor; ccRCC, clear cell renal carcinoma; CML, chronic myeloid leukemia; CNV, copy number variation; CRC, colorectal cancer; CTC, circulating tumor
cells; CyTOF, cytometry by time of flight; ETMR, embryonal tumors with multilayered rosettes; FACS, fluorescence-activated cell sorting; GBM,
glioblastoma multiforme; HCC, hepatocellular carcinoma; HNSCC, head and neck squamous cacrinoma; MACS, magnetic-activated cell sorting; MARS-seq,
massively parallel RNA single-cell sequencing; MM, metastatic melanoma; NSCLC, non-small cell lung cancer; NSCL ADC, non-small-cell lung
adenocarcinoma; PC, prostate cancer; PDX, patient-derived xenograft; RCC, renal cell carcinoma; SCC, squamous cell carcinoma; SNV, single-nucleotide
variant; TCR, T cell receptor; TCR-seq, T cell receptor sequencing; UM, uveal melanoma; WGA, Whole-genome amplification; WNT MB, WNT-subtype
medulloblastoma.

multiplex protein analysis,166 and to profile mRNA and protein simul-
taneously in colorectal tumor tissue.167 While this approach allows for
combined multiplex mRNA and protein analysis, the GeoMx/DSP plat-
form lacks single-cell resolution and requires a priori knowledge of
target protein markers or mRNAs together with reliable markers to
visualize tissue structure. Interestingly, GeoMx-based analysis of B
cells was complemented by technologies assessing mRNA and surface
proteins at single-cell resolution.166
In contrast, high-definition spatial transcriptomics (HDST) enables
unbiased mRNA profiling with 49% of the spatial barcodes being
assigned to a single-cell type and was successfully used to distinguish
cell types in breast cancer,160 while offering greater spatial resolution
compared to similar approaches.168,169 Combining spatial trans-
criptomics with conventional high throughput single-cell sequencing
enabled more refined spatial cell type annotations in pancreatic ductal
ADC.170 Despite these promises in spatial transcriptomics, future
developments need to improve the current sparsity of HDST and to
demonstrate compatibility of this method with Formalin-Fixed
Paraffin-Embedded (FFPE) sections, which represent the dominant
form in which solid tumor specimen are preserved to date. Interest-
ingly, the commercially available Visium chemistry (10X Genomics)
has recently been applied successfully to FFPE sections of the mouse
brain and ovarian carcinosarcoma as exemplified by a study currently
available as a preprint,171 opening up for the possibility to perform
spatial transcriptome analysis on clinical FFPE samples.
Alternatively, highly specific in-situ hybridization (RNAscope)
157
enables detection of mRNA molecules in FFPE tissue and was used
successfully for automated, quantitative profiling of HER2 status in
breast carcinoma.172 While providing cellular and subcellular resolu-
tion, a priori knowledge of targets is necessary and highly multiplexed
tissue analysis is currently not possible. However, a higher degree of
multiplexed targeted gene mRNA detection in breast cancer has been
achieved using padlock sequencing.173 Expansion of RNAscope by the
usage of oligonucleotides conjugated to metal-chelated reporters to
bind RNA-probes during the final hybridization step facilitates simul-
taneous labeling of protein structures using metal-conjugated anti-
bodies. This in turn enables simultaneous profiling of mRNA and
proteins from the same section using IMC and was shown to success-
fully correlate with mRNA and protein expression levels in a large
cohort of samples providing architectural maps of breast cancer tissue
at spatial single-cell resolution.161
In addition to spatially resolved mRNA and protein expression,
recent technological advances linked the genomic profile of a single-
cell to its position in a given tissue.162,163 Similar to both approaches
is that the tumor tissue is subjected to hematoxylin & eosin (H&E)
staining to visualize structural elements, followed by subsequent isola-
tion of single-cells using UV laser162 or isolation of groups of cells
down to single-cells using single infra-red (IR) pulses.163 This made it
possible to spatially resolve genomic aberrations occurring during an
early stage tumor such as ductal breast carcinoma,162 and holds great
potential to increase our understanding of how tumor infiltration and
invasion processes occur at the single-cell level in the context of the
tumor microenvironment.
Taken together, a broad variety of technologies allowing single-
cell readouts in a spatial context are available, which have offered
highly relevant insights by integrating different modalities, such as
mRNA and protein expression or genomic aberrations within the
tissue context. These approaches differ in their capacity of cellular
throughput, compatibility with clinical samples, single-cell resolu-
tion, preservation of tissue integrity for downstream analyses, the
degree of multiplexed detection, and the necessity of a priori
knowledge on tissue-specific targets. Therefore, selection of a
methodology for spatial tissue analysis often necessitates a
compromise with regard to several aspects. For example, FFPE-
compatible mRNA analysis at single-cell analysis requires a trade-
off in the number of transcripts, which can be processed at the
same time.
Overall, spatial tissue analysis at single-cell resolution still needs
to overcome several limitations in order to become a widely used clin-
ical diagnostic technology but given the rapid development and dem-
onstrated high promise of this technology it is likely that we will
witness significant advancement toward clinical applicability in the
near future.
7 | C HA LLE NGE S FO R C LI NI C A L
TRANSLATION OF SINGLE-CELL
SEQUENCING
As described in previous sections, not only rapid technological pro-
gress but also the potential of analyzing tumor tissue routinely at
single-cell resolution has now become feasible in a research setting.
Single-cell analyses in cancer has opened up for the possibility to
putatively aid diagnostics,148,172 to monitor treatment
72,76,85,87,149
response or to refine treatment processes59-61,72 toward
personalized therapies and has thus spurred great interest to translate
such technologies into routine clinical applications.
Single-cell analyses regardless of modality or spatial resolution,
currently requires cost intensive, large-scale sequencing reactions in
order to process a clinically informative cohort of patient samples and

15
to extract sufficient numbers of single-cells to guarantee statistically
valid analyses to infer reliable diagnostic information. Novel strategies
for multiplexing single-cell transcriptomes50,51 or single-cell
133
genomes have greatly increased the possible cellular throughput
per sample. However, in particular WES and WGS of single-cells for
de novo SNV calling necessitate high sequencing depths, rendering
these approaches currently economically challenging for large-scale
studies. Additionally, increasing single-cell throughput together with
multiomic readouts and higher-dimensional data require sophisticated
expertise for data analysis in addition to large computational infra-
structure. Approaches of combined low and high coverage
analyses,130,141,147 targeted qPCR-based analyses153,174 and more
recently microfluidic droplet-based analysis149,175 present more cost
effective alternatives to assess genomic aberration in cancer.
Alternatively, inference of genomic alterations from less cost
intensive mRNA-sequencing strategies may provide an attractive
strategy to facilitate introduction of single-cell sequencing in a clinical
diagnostic setting.35,37,38,71,73,74,76 However, such approaches provide
only indirect genomic information, which is furthermore limited due
to the necessity that a genomic lesion needs to be manifested on the
mRNA level which on top of that can be captured by the chosen
single-cell chemistry. Also, most solid tumor samples are stored as
FFPE tissue blocks which often yield low quality mRNA thus render-
ing single-cell transcriptomics challenging.
While Smart-seq2-based full-length mRNA-sequencing at single-
cell resolution generates more transcriptional information which can
be used to infer genomic aberrations, its analysis cost per cell on
sorted plates amounts to approximately 30 USD as opposed to 4 USD
on commercial platforms such as the iCell8cx. In contrast, droplet-
based mRNA-sequencing on the 10X Chromium provides a more cost
effective alternative and processing cost of 0.5USD per cell. For fur-
ther comparison of different platforms, methodologies, cohort size in
selected reference literature see Tables 2 and 3.
Initially, single-cell studies focused on manual isolation of indi-
128,129
vidual cells, an approach that does not meet the required cellu-
lar throughput for clinical applications. Technological advancements
such as the use of microwells43,49 and nanodroplets44-46 have greatly
increased the cellular throughput, however, such methods require
rather large amounts of starting cell numbers and provide compara-
bly low capturing rates,45,46 thereby rendering these technologies
less favorable in a clinical setting when sample size may be small and
limiting. The necessity to enrich for distinct cell populations via anti-
body staining and flow cytometry provides another example of sam-
ple loss, which becomes particularly unfavorable when analyzing
60,61,146-148
low input samples and rare cell types such as CTCs. In
order to generate clinically valuable results, capturing of extremely
rare clones in a tumor sample must be guaranteed which in turn
requires processing of patient-derived samples in their entirety
agnostic of sample size. Platforms to isolate scarce clinical samples
for high throughput analysis, for example, the sciFLEXARRAYER S3
or cellenONE systems, are becoming available and have recently
been used successfully for low-coverage CNV profiling of human
breast cancer samples.137
16
Another important obstacle in order to achieve clinical translation
of single-cell technologies is sample comparability with regards to sam-
ple isolation, molecular characterization and downstream computational
analyses. It is known, that cell dissociation strategies can alter transcrip-
tional signatures155 and that cell isolation needs to be evaluated care-
fully prior to starting an experiment.176 Furthermore, it has been shown
that gene expression patterns are induced which are distinct for
biopsy- and autopsy-derived brain samples177 and which biases down-
stream analyses. Thus, the effect of sample isolation, storage and sam-
ple type on the modality analyzed (mRNA, DNA, protein) needs to be
systematically assessed in order to define common classifiers to facili-
tate comparability of results between different research centers and
across a large space of clinical samples. Integration of heterogeneous
data sets obtained at different research centers using different method-
ologies will require advanced batch correction24 and computational
tools to combine these data sets in a meaningful way while minimizing
overcorrection and maintaining relevant biological differences.178
Extensive benchmarking of single-cell technologies179 together
with interrogation of sampling artefacts180 are pivotal in order to
achieve overall comparability of test results. A recent study has carried
out a systematic comparison of different cell and nuclei isolation strate-
gies on a diverse range of clinical cancer samples. Testing several isola-
tion protocols per sample type, the authors based their evaluation
among other metrics on the cellular diversity a given protocol would
reproduce. Taken together, this study provides an extensive resource
suggesting highly specified isolation protocols for various cancer tissues
with the overarching goal to provide standardizable, robust and compa-
rable single-cell workflows for the use in a clinical setting.181
The results of an interrogation of clinical samples by single-cell
analysis may vary depending on the platform used. It is therefore cru-
cial to systematically assess existing platforms for single-cell genome
and spatial tissue analysis, similarly to single-cell transcriptome
chemistries,54,55 to determine most suitable applications and experi-
mental conditions for defined clinical questions.
With regards to mRNA analysis, definition of a statistically critical
number of cells for computational analyses, optimal sequencing depth in
addition to the minimum number of cells necessary to define a cell type
or state, among others, will be crucial for improved comparability. In order
to provide sequencing data, which allow for comparable mutation analysis
and variant calling, a unified way to define clonal structures will need to
be established.134 Novel computational tools are required to manage data
analysis in increasingly large data sets, as has been demonstrated previ-
ously.52,178 Computational challenges associated with the analysis of can-
cer samples by single-cell transcriptomics are reviewed elsewhere.26 In
addition, analysis of longitudinal clinical samples is likely to provide insight
into disease progression and treatment response.175 In order to relate lon-
gitudinal samples, existing trajectory inference methods182,183 need to be
developed further to integrate different modalities of the same sample
along a pseudo-time axis, to resolve multiple clonal subtypes and to per-
form high-dimensional comparative analyses against large patient cohorts
while maintaining the longitudinal order of cell states and clones.
Moreover, to reach clinical validity, existing single-cell methodolo-
gies need to possess optimal detection sensitivity and specificity. The
T A B L E 3 Overview of technical details of translational research articles highlighted in this review, which describe genomic, epigenetic, or
spatial analyses

17
Abbreviations: ADC, adenocarcinoma; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukaemia; ATAC, assay for transposase-accessible
chromatin; BCC, basal cell carcinoma; CITEseq, cellular indexing of transcriptomes and epitopes by sequencing; CLL, chronic lymphocytic leukemia; CNA,
copy number alteration; CNV, copy number variation; DSP, digital spatial profiler; FACS, fluorescence-activated cell sorting; FL, follicular lymphoma; HDST,
high density spatial transcriptomics; HCC, hepatocellular carcinoma; MPAL, mixed-phenotype acute leukemia; MRD AML, minimal residual disease acute
myeloid leukemia; PC, protstate cancer; PDAC, pancreatic ductal adenocarcinoma; PHLI-seq, phenotype-based high-throughput laser-aided isolation and
sequencing; QRP DOP-PCR, quasi-random priming degenerate oligonucleotide primed polymerase chain reaction; RCC, renal cell carcinoma; SNV, single-
nucleotide variant; SCLC, small cell lung cancer; SS, synovial sacroma; ST, spatial transcriptomics; TNBC, triple-negative breast cancer; WES, whole-exome
sequencing; WGS, whole-genome sequencing.

F I G U R E 3 Timeline illustrating key references utilizing single-cell technology in translational cancer research for transcriptome analysis and
immune profiling. Chronological appearance of selected references highlighted in this review in which RNA analysis and immune profiling in the
context of various different cancer types were performed. At the bottom, schematic illustration of key platforms used over time to isolate and
process single cells such as FACS isolation into PCR plates, microfluidic devices such as the Fluidigm C1, droplet-based technologies such as 10X
Genomics, laser-catapulting, imaging mass cytometry and spatially resolved analysis of the transcriptome. AML = acute myeloid leukemia;
ATRT = atypical teratoid/rhabdoid tumors; CML = chronic myeloid leukemia; CTCs = circulating tumor cells; CyTOF = cytometry by time of flight;
ETMR = embryonal tumors with multilayered rosettes; FACS = fluorescence-activated cell sorting; GBM = glioblastoma multiforme;
HNSCC = head and neck squamous carcinoma; MARS-seq = massively parallel RNA single-cell sequencing; PDX = patient-derived xenograft;
TCR-seq = T cell receptor sequencing; WGA = whole-genome amplification; WNT MB = WNT-subtype medulloblastoma (n.a.: not available/
disclosed in article)

18
F I G U R E 4 Timeline illustrating key references utilizing single-cell technology to study epigenetic alterations and genomic aberrations in single
cancer cells as well as selected references studying cancer tissues with spatial resolution. Chronological appearance of selected research articles
highlighted in this review assessing epigenetic and DNA changes in cancer samples and the progressive emergence of studies spatially resolving
different modalities such as mRNA and protein expression as well as genomic alterations in cancer tissue. Schematic representations of key
platforms frequently used in various different single-cell studies such as FACS isolation into PCR plates, microfluidic devices such as the Fluidigm
C1, droplet-based technologies such as 10X Genomics, laser-catapulting, imaging mass cytometry and spatially resolved analysis of the
transcriptome. ALL = acute lymphoblastic leukaemia; AML = acute myeloid leukemia; ATAC-seq = assay for transposase-accessible chromatin;
CITE-seq = cellular indexing of transcriptomes and epitopes by sequencing; CLL = chronic lymphocytic leukemia; CML = chronic myeloid
leukemia; DOP PCR = degenerate oligonucleotide primed polymerase chain reaction; FACS = fluorescence-activated cell sorting; GeoMx
DSP = GeoMX Digital spatial profiler; GS = genome sequencing; HDST = high density spatial transcriptomics; IP = immunoprecipitation;
MALBAC = multiple annealing and looping based amplification cycles; MDA = multiple displacement amplification; MRD AML = minimal residual
disease AML; PDAC = pancreatic ductal adenocarcinoma; QRP DOP-PCR = quasi-random priming DOP-PCR; PHLI-seq = phenotype-based high-
throughput laser-aided isolation and sequencing; RCA = rolling circle amplification; SCLC = small cell lung cancer; ST = spatial transcriptomics;
TNBC = triple-negative breast cancer; Trio-seq = triple omics sequencing; WES = whole-exome sequencing; WGA = whole-genome amplification
(n.a.: not available/disclosed in article)

minute starting amounts of mRNA or DNA in single-cells often require as dropout events resulting from transcripts which were not captured
extensive amplification prior to sequencing which introduces technical during reverse transcription.184 Such noise impairs overall detection
134
noise such as allelic dropouts or polymerase-induced errors, as well confidence. Both continuous improvement of sample preparation and

19
computational models are required to correct for such errors for any
platform and chemistry which is intended to be used in a clinical set-
184,185
ting, as exemplified here for single-cell mRNA-sequencing.
Advances in this field will facilitate not only the cellular
deconvolution of cancer tissues but also to build cancer-specific clas-
sifiers based on several modalities, thereby refining current cancer
classification and treatment of cancer. Ultimately, single-cell data of
distinct modalities will need to be put into the context of the tumor
tissue, where transcriptomic and genomic signatures are translated
into altered functionality of cells in the diseased state.
8 | C O N CL U S I O N S
The tremendous technological development in single-cell sequencing of
the past decade has yielded a broad toolbox to study many modalities in
cancer, such as mRNA,37,38,57,59 DNA alterations,141,144,146-149,154,175
immune cell composition of tumors,85-87,90,92,95 chromatin
changes 116-118,122 97
and metabolic effectors in dissociated single-cells or
nuclei as well as within the context of diseased tissue156-164,167,172
(Figures 3 and 4). Mono-, bi-, or even-multimodal approaches have
within short time facilitated cancer research at unprecedented depth and
gained invaluable information on tumor composition and classification,
clonal evolution in cancer, disease progression and treatment response.
Nevertheless, many methods fall short in providing information
on tissue context, which can provide further information of prognostic
value.80,186 The achievement of clinical translation of spatially
resolved methodologies depends, among others, on the ability to com-
prehensively analyse high dimensional data comprised of information
on cell type and state, cell boundaries to adjacent cells together with
the cells location within the tissue. This in turn requires the develop-
ment of computational tools which allow for robust identification of
given patterns of cells in a tissue or tissue motifs,187 which then may
enable for in silico construction of tissue network structures and to
ultimately infer pathological processes, necessary to classify patients
and to aid diagnosis.
While current technical limitations prevent broad clinical application
of the aforementioned methodologies, it seems clear that single-cell ana-
lyses will become an integral part in clinical diagnostics, prognostication,
disease follow-up, and treatment selection in the next coming years. This
is strongly emphasized by the large number of studies and their diverse
scope employing transcriptome-sequencing of single cells (Figure 3). In
addition, existing single-cell DNA applications often present sufficient
analytical validity and additional refinement regarding detection sensitivity
and specificity of those methods may ultimately render bulk WGS/WES
obsolete, which are currently often used to either substantiate findings
obtained via single-cell sequencing144 or to nominate genomic lesions for
targeted single-cell analysis.149,154,175 Further, each existing technology
possesses specific opportunities but also technical shortcomings, which
will affect their analytical validity and which will therefore lead to varying
time frames for clinical translation. Nevertheless, the literature highlighted
in this review clearly demonstrates the applicability and usefulness of
single-cell analysis in cancer research and diagnostics.
20
CONFLIC T OF INT ER E ST
The authors declare no potential conflict of interest.
DATA AVAILABILITY STAT EMEN T
Data sharing not applicable to this article as no datasets were gener-
ated or analysed during the current study.
OR CID
Ulrich Pfisterer https://orcid.org/0000-0002-4613-6427
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25
 Identification of a tumor–specific gene
regulatory network in human B-cell lymphoma

Introduction
Simultaneous readout of transcriptomic and epigenomic Highlights
data from the same cell at single cell resolution allows for
• Distinguish tumor versus normal cells in
direct reconstruction of cell type–specific gene regulatory
a heterogeneous sample
networks that does not rely on inference or assumptions to
tie the two data types together. Here, we show how multio- • Reconstruct cell type–specific gene regulatory
mic analysis of paired RNA-seq and ATAC-seq data from network
the same single cells using Chromium Single Cell Multi- • Confirm PAX5 as a critical regulator specific
ome ATAC + Gene Expression enables direct linkage of to tumor B cells
differentially accessible DNA regions to proximal differen-
• Identify putative target genes downstream
tially expressed genes to identify putative regulatory
of PAX5
targets. As a result, you can answer questions not only
about what genes are expressed in a single cell, but how
expression is regulated through associated open chroma-
tin regions. In a diffuse small B-cell lymphoma sample, we
confirmed Paired Box 5 (PAX5) as an important regulator
in tumor B cells and identified a network of potential
PAX5 target genes.

Sample prep GEM generation Library construction Sequencing Data processing Data visualization

Prepare
Nuclei
Suspension

Figure 1. Experimental methods for nuclei isolation and multiomic data generation. Flash-frozen intra-abdominal lymph
node tumor, with pathologist annotation of diffuse small B-cell lymphoma tissue, was acquired from BioIVT Asterand®.
Nuclei were isolated following the Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression
Sequencing Demonstrated Protocol (CG000375). Isolated nuclei were flow sorted before permeabilization. Nuclei were
transposed in bulk before single nuclei encapsulation in GEMs (Gel Bead-in-emulsion), where DNA fragments and the 3’
ends of mRNA were barcoded. Paired ATAC and gene expression libraries were generated from 14,000 total nuclei as
described in the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338 Rev A) and
sequenced on an Illumina NovaSeq™ 6000 v1.5.

26
 B
Tumor B cycling
pDC
Cell type
B
Fibroblasts
Mono
Stromal cells Mono
pDC

umap2

umap2
Stromal cells Fibroblasts
T
pDC T T cycling
Mono T cycling
Tumor B
Tumor B cycling
Tumor B cycling
NA
Fibroblasts Tumor B

Stromal cells

A. Gene expression
Gene Expression
Gene Expression ATAC
ATAC
ATAC
Gene expression, T cells
Gene Expression, T cells
umap1

ATAC, T cells
uma

B B
Tumor B Tumor B Treg NKT

B B
Tumor B cycling
Tumor B cycling CD4 memory
pDC pDC T cell subtype
Cell type Cell type Cell type Cell type CD8 Naive
CD8 memory CD4 cytotoxic
B B B B
CD4 Naive CD4 memory
Fibroblasts Fibroblasts Fibroblasts Fibroblasts
CD4 Naive
Mono Mono Mono Mono
Stromal cells
Stromal cells Mono Mono CD4 Tfh
pDC pDC pDC pDC
umap22

umap22

umap22
umap2
umap2

umap2
T cycling T cycling Proliferating T CD8 Exhausted
UMAP

UMAP

UMAP
Stromal cells
Stromal cells Fibroblasts
Fibroblasts Stromal cells
Stromal cells
NK CD8 memory CD
T T T T
CD8 Naive
pDC pDC T T T cycling T cycling T cycling T cycling CD4 Tfh
Mono Mono T cycling T cycling NK
Tumor B Tumor B Tumor B Tumor B
T T NKT
Tumor B cycling
Tumor B cycling Tumor B cycling
Tumor B cycling CD8 Exhausted
Tumor B cycling
Tumor B cycling Proliferating T
NA NA NA NA
Fibroblasts
Fibroblasts Tumor B Tumor B CD4 cytotoxic Treg

Stromal cells
Stromal cells

umap1
UMAP 1 umap1 umap1 umap1
UMAP 1 umap1
UMAP 1 uma

B. MS4A1 BANK1 PAX5

Gene Expression, T cells T cells
Gene Expression, ATAC, TATAC,
cells T cells

Tumor B cells
Treg TregNKT NKT

CD4 memory
CD4 memory
T cell subtype
T cell subtype T cell subtype
T cell subtype
CD8 Naive
CD8 Naive CD8 memory
CD8 memory CD4 cytotoxic
CD4 cytotoxic CD4 cytotoxic
CD4 cytotoxic

CD4 Naive
CD4 Naive CD4 memory
CD4 memory CD4 memory
CD4 memory
umap2 UMAP 2

umap2 UMAP 2

UMAP 2
CD4 Naive CD4 Naive CD4 Naive CD4 Naive
CD4 Tfh CD4 Tfh CD4 Tfh CD4 Tfh
umap2
umap2

Proliferating T
Proliferating T CD8 Exhausted
CD8 Exhausted Proliferating T
Proliferating T CD8 Exhausted
CD8 Exhausted
NK NK CD8 memory
CD8 memory CD8 Exhausted CD4 Tfh CD4 Tfh
CD8 Exhausted CD8 memory
CD8 memory
CD8 Naive CD8 Naive CD4 cytotoxic
CD4 cytotoxic CD8 Naive CD8 Naive
CD4 Tfh CD4 Tfh
NK NK NK NK
Treg Treg
NKT NKT NKT NKT
CD8 Exhausted
CD8 Exhausted CD8 Naive
CD8 Naive
ProliferatingProliferating
T T ProliferatingProliferating
T T
CD4 Naive
CD4 Naive
CD4 cytotoxic
UMAPCD41 cytotoxic Treg Treg UMAP 1 CD4 memory
CD4 memory Treg Treg UMAP 1

CD8 memory
CD8 memory

Figure 2. Simultaneous measurement of gene expression and open chromatin profiles from the same single nuclei enables NKT NKT

clustering based on either modality. A. Shown are clustering and manual annotation based on gene expression for all 14,000
NK NK

nuclei (left); gene expression-derived annotations layered on ATAC projections (middle); and the gene expression plot on the
umap1 umap1 umap1 umap1

left restricted to the T-cell populations (right). B. Highlighted are expression levels of select genes, including MS4A1, a canonical
B-cell marker (left); BANK1, an attenuator of BCR activation pathway that is repressed in tumor cells relative to normal B cells
(middle); and PAX5, required for B-cell differentiation (right).

A. B.
Feature linkage Annotate peaks linked to DEGs

Figure 3. Computational strategy for identification of cell type–specific gene regulatory networks. A. In 10x Genomics Cell
Ranger ARC software, feature linkages are defined as pairs of genomic features, such as peaks and genes, that exhibit signifi-
cant correlation in their chromatin accessibility and transcript level, respectively, across cells. Feature linkages can be positively
or negatively correlated. For example, an open enhancer region may have a positive correlation with gene expression of its
associated transcript (blue), while the binding of a repressor would result in a negatively correlated feature linkage (red). The
greater the correlation between open chromatin signal and gene expression, the taller the arc. B. To identify a gene regulatory
network in tumor B cells, genes were first filtered based on significant transcriptional upregulation in tumor B cells relative to
normal B cells (p < 10 -20), resulting in 198 differentially expressed genes (DEGs, green). Peaks associated with DEGs (green) were
identified using feature linkages. Tumor B cell–specific enriched motifs were then identified using DEG-linked peaks. Enriched
motifs and linked upregulated genes were used to define a B cell lymphoma–specific gene regulatory network (Figure 4).

27
 What to look for
Since mRNA and ATAC data are generated from the same
cells, cell-type annotations can be transferred from one
modality to the other (Figure 2A, middle). In addition to
the identification of B cells, monocytes, and T-cell sub-
types using canonical cell markers like the B-cell marker
MS4A1, tumor B cells were distinguishable from normal
B cells based on upregulated CD40 expression (data not
shown) and reduced BANK1 (Figure 2B). PAX5 was sig-
nificantly upregulated in tumor B cells relative to normal
B cells (Figure 2B), and has previously been identified as
a core regulator of chronic lymphocytic leukemia (CLL)
(Ott et al., 2018).
Paired gene expression and open chromatin signals pave
the way for high-confidence gene regulatory network pre-
dictions using feature linkages, which are calculated
automatically in Cell Ranger ARC (Figure 3A). Feature
linkages help build putative gene regulatory networks by
providing correlated gene expression and open chromatin
regions across the genome. To identify tumor B cell–spe-
cific gene regulatory networks, we first annotated feature
linkages by genes upregulated in tumor B cells to identify
peaks that were potential drivers of differential expres-
sion. We then identified motifs enriched in these peaks
relative to a set of matched background motifs within
tumor B cells (Figure 3B). Using this method, we found
that the PAX1 motif was the most enriched (Figure 4).
PAX1 and PAX5 motifs are highly similar, however PAX1
is not expressed in tumor B cells, while PAX5 is highly
expressed (Figure 4). Therefore, it is likely the PAX5 tran-
scription factor is binding the identified PAX1 motif. This
inference is only possible with paired gene expression
and open chromatin information from the same cells.
To understand the role of PAX5 in tumor B cells, we
zoomed in on the PAX5 locus, which is differentially
expressed between B cells and tumor B cells (Figure 5).
Expression of PAX5 is highly correlated with open PAX5
motif sites in a previously identified super-enhancer, sug-
gesting autoregulation (Figure 5, dashed box). Additional
feature linkages contribute further to the reconstruction
of a putative tumor B cell–specific gene regulatory net-
work, and suggest PAX5 may also regulate the immune
transcription factor genes NFATC1, TCF4, IKZF1, and
IRF8 (Figure 4). The importance of PAX5 and its position
as a key genetic regulator in tumor B cells is consistent
with previously published results showing that, of 147
transcription factors tested, loss of PAX5 had the great-
est effect on cell proliferation in a CLL cell line (Ott et
al., 2018). While confirmation of individual links in our
predicted gene regulatory network requires functional
tests, the confidence in regulatory connections is greatly
increased by joint measurement of mRNA and ATAC data.

Target genes

Immune TFs Other Immune Genes Linkage

Significance
PAX5 200
ONECUT1 100
MOTIFS PAX1 50
CUX1 25
PAX9
CUX2 10
0

TC
FOF4
N XP1
PA TC
IK 5
AH 1
TOR
IR
PO 8
TP U2
LE 3 2
C
TF RD
ST C 1
BC GA
D L2 1
SKX1
C AP2
SY 83
IL K
R R
C SG
D KN P3
KL 1 A
BL L6
SE K
PA MA
IG 2 A
AD 1
N TR
FCKBI
FCRL3

enrichment log10 UMI

FA

A
D R
LG 2

F P
4
ZF

LC
X 1

K 4
F1

R 1

H
N
6 F

6
X

R
L2
Z
L

Figure 4. Feature linkages help build a tumor-specific gene regulatory network. The table summarizes significant feature
linkages between motifs in the PAX/CUX/ONECUT family and a selection of immune-related transcription factors (TFs) and
other immune genes that are differentially expressed in tumor B cells. At far left, the blue line plot shows motif enrichment
scores, calculated using the analysis outlined in Figure 3. Gene expression levels of the transcription factors expected to bind
each motif are indicated in the adjacent bar graph. For every differentially expressed gene–PAX/CUX/ONECUT motif pair, the
significance of the most significant feature linkage is indicated by a colored square.

28
Figure 5. Loupe Browser enables visualization of feature linkages. Positively correlated feature linkages are denoted by
arcs at top. Highlighted by the dotted box is a highly significant feature linkage between PAX5 and a previously annotated
CLL super-enhancer that is depicted in black (Ott et al., 2018). Below the illustrated feature linkages are open chromatin peaks
identified for each cell cluster across a 0.3 Mb region. Annotated cell types are color coded. On the right are plots showing the
expression level of PAX5 (top) and accessibility of the linked super-enhancer (bottom) for each annotated cell type. Tumor
B cells (blue), in contrast to normal B cells (red), have elevated PAX5 expression and open chromatin at this super-enhancer.

Explore what you can do Resources

Chromium Single Cell Multiome ATAC + Gene Expression To explore the dataset further, download the data here:
helps you identify the critical regulators and pathways https://support.10xgenomics.com/single-cell-multiome-
behind cell state. Putative gene regulatory networks can atac-gex/datasets/1.0.0/lymph_node_lymphoma_14k
be built based on correlated gene expression and open
chromatin sites with greater accuracy and confidence
than would be possible with a single modality. At the
References
same time, the identity of likely transcriptional regula- Ott CJ, et al. Enhancer Architecture and Essential Core
tors can be constrained by both expression level and Regulatory Circuitry of Chronic Lymphocytic Leukemia.
motif availability. Multiomic readout at the transcrip- Cancer Cell. 34: 982–995, 2018.
tional and epigenetic levels, particularly from the same
single cell, takes much of the guesswork out of network
reconstruction based on gene expression alone, enabling
a deeper understanding of the molecular mechanisms
underpinning disease progression, developmental differ-
entiation, and therapeutic response.

Contact us
10xgenomics.com | info@10xgenomics.com
© 2021 10x Genomics, Inc. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
LIT000110 - Rev A - Data Spotlight - Tumor–specific gene regulatory network in human B-cell lymphoma

29
 SPECIAL FEATURE REVIEW

Recent advances in single-cell multimodal analysis to study

immune cells
Raymond HY Louie & Fabio Luciani
School of Medical Sciences, The Kirby Institute, University of New South Wales (UNSW), Sydney, NSW, Australia

Keywords
Abstract
cell state, cell–cell interaction, clonal analysis,
immune cells, lineage, multimodal analysis, Recent advances in single-cell technologies have enabled the profiling of the
pseudotime, single-cell technology, temporal genome, epigenome, transcriptome and proteome, along with temporal and
analysis
spatial information of individual cells. These technologies have provided
unique opportunities to understand mechanisms underpinning the immune
Correspondence
system, such as characterizations of the molecular cell state, how the cell state
Fabio Luciani, School of Medical Sciences and
the Kirby Institute, University of New South
evolves along its lineage and the impact of spatial location on cell state. In this
Wales (UNSW), Sydney, NSW 2052, review, we discuss how these mechanisms have been studied through recent
Australia. advances in single-cell multimodal technologies.
E-mail: luciani@unsw.edu.au

Received 5 September 2020; Revised 30

October, 24 November and 9 December
2020; Accepted 9 December 2020

doi: 10.1111/imcb.12432

Immunology & Cell Biology 2021; 99:

157–167

different stages of cellular differentiation. This can be

INTRODUCTION
Recent advances in single-cell technology has made it
possible to simultaneously extract different types of
information, or “modalities,” from the same single cell.
These modalities can arise from the genome, epigenome,
transcriptome and proteome (Figure 1a). In each of these
ome-layers, different modalities exist, such as mutations
and copy number variations at the genome layer, DNA
methylation and chromatin accessibility at the epigenome
layer and unspliced and spliced messenger RNA (mRNA)
at the transcriptome layer. Single-cell multimodal analysis
has been used for different immunological applications.
For example, a common application is to characterize the
molecular cell state, which can be described by a single
modality, for example, the expression of certain genes, or
by a combination of modalities spanning across the
genome, epigenome, transcriptome and proteome
(Figure 1a).
Single-cell multimodal analysis can also be used to
describe how the molecular cell state evolves along
achieved through combining “ome-layers” and temporal
modalities, thus capturing the information related to the
ordering of cells at different stages of differentiation.
Combining ome-layer and temporal modalities can
characterize how the molecular state of an immune cell
evolves along its lineage, from hematopoietic stem cells
(HSCs) in the bone marrow to its cell fate. A cell’s
lineage is created by the developmental history of the cell,
with each cell belonging to the same or sister clones.
Single-cell multimodal analysis can also inform on how
molecular cell state is location dependent, which requires
spatial modalities. These modalities include the (1) spatial
location of the cell in the body or (2) which cells are
interacting with each other. Cell-to-cell interactions can
be determined by examining receptor–ligand pair
interactions and are important, as neighboring cells can
modulate cell function through these interactions.
Single-cell multiple modalities can be obtained either
experimentally or bioinformatically. Experimental
modalities can be obtained through gathering cytometric

30
 (a)

Intracellular
Genome
proteome
DNA mutations

Surface proteome

Transcriptome Epigenome
RNA chromatin
accessibility,
methylation

(b) Molecular cell state

Heterogeneous population

Mode 1 Mode 2

Single-cell multi-modal applications

Temporal Spatial
(e.g., gene, protein)
Marker expression

Location in body
Cell-cell interaction

Time

Figure 1. Single-cell multimodal analysis: components and applications. (a) Cellular components of single-cell multimodal analysis. (b)
Applications of single-cell multimodal analysis to molecular cell state, temporal evolution and spatial analysis.

information before a destructive assay, separation of
cellular components or a conversion of cellular
information into a common molecular format.1 Detailed
descriptions of these methods are given in an excellent
review,1 and will not be discussed further. Bioinformatic
tools can also be used to extract multiple modalities. The
key difference from the experimental approach is that
these modes are extracted from data generated from the
same assay, where the data are typically sequenced reads.
For example, at the transcriptomics layer, reads aligned
to a transcriptome can be processed bioinformatically to
yield unspliced and spliced RNA.2 Data obtained at one
layer can also be used to computationally predict
modalities at another layer. For example, transcriptomic
data have been used to predict cell-to-cell interactions at
the spatial layer,3,4 the temporal ordering of cells5,6 and
the future states of cells2 at the temporal layer.
In this review, we will discuss recent single-cell
multimodal applications to human or mouse immune
cells, in contrast to broad reviews which focus on general
applications of single-cell multimodal analysis.7 We
define single-cell multimodal analysis as the analysis of
data sets arising from at least two modalities obtained
from the same cell, as opposed to bioinformatically

31
 integrated data sets arising from different samples
(addressed in previous reviews1). As summarized in
Table 1, we will review recent advances demonstrating
the impact of single-cell multimodal analysis in
understanding molecular cell state, temporal and spatial
location of immune cells (Figure 1b). Finally, we will
discuss future research opportunities.
MOLECULAR CELL STATE
The molecular state of an immune cell can be
characterized by a combination of modalities from the
genome, epigenome, transcriptome and proteome
(Figure 1a). A common application of multimodal
information is to isolate cells with a certain state using
one modality, and then examine the cell state of these
isolated cells in another modality. This process is
sometimes repeated multiple times at different modalities.
For example, surface protein markers have been
traditionally used to first isolate or sort cells by
fluorescence-activated cell sorting, followed by analysis
using gene expression, immune receptors, chromatin
accessibility regions or combinations of these modalities.
One of the key advantages of single-cell analysis is to
dissect the cellular and molecular heterogeneity in a
tissue or sample, and even to identify subsets within the
same cell type. The identification of cell states using
multimodal analysis has been applied to analyze immune
cells in healthy and disease, pathogen infection,
autoimmune and cancer samples.
Identification of cell state in healthy samples
Single-cell multimodal analysis can be used to isolate cell
subsets and characterize their molecular signatures from
healthy samples, which can then be used as a baseline
reference when comparing with immune cells from
disease samples. For example, a recent study explored T-
cell composition in lymphoid and nonlymphoid tissues
from both healthy humans and mice.8 By combining
single-cell gene expression with T-cell receptor (TCR)
sequences, this study showed distinctive signatures
between regulatory and memory subsets across lymphoid
and nonlymphoid tissues, and also similar subsets of
regulatory T cells across humans and mice. Unexpectedly,
this integrated analysis also revealed that the same T-cell
clones (i.e. with identical TCR) could be identified in
lymphoid and nonlymphoid samples, thus suggesting
migration of regulatory cells between organs.
Single cells can be separated using high-purity
fluorescence-activated cell sorting into wells, after which
mRNA or DNA is extracted for single-cell analyses. This
is the case for plate-based approaches such as Smart-seq2
32
or similar protocols, as previously reviewed.9 However,
these methods are laborious and only applicable for small
cell number. New methods are available to isolate single
cells at high throughput, for instance, utilizing cellular
bar codes and unique molecular identifiers [e.g.
microfluidics technology (10x Chromium) or nano plates
(Rhapsody)].9 These methods require demultiplexing of
individual cells which is performed bioinformatically.
While these approaches were first developed to perform
single-cell RNA sequencing (scRNA-seq), more recently
these have been also developed to perform multimodal
analyses. For example, Cellular Indexing of
Transcriptomes and Epitopes by Sequencing (CITE-Seq)10
and AbSeq11 are two technologies which can
simultaneously extract intracellular (surface) protein and
gene expression in the same cell. These technologies have
been used to explore heterogeneous populations in both
healthy and disease samples.10–12 For example, CITE-
Seq10 was applied in combination with 10x Chromium to
identify cord blood mononuclear cells and successfully
identify natural killer cells based on the CD16 and CD56
surface markers, after which gene expression analysis
revealed differentially expressed signatures of natural
killer subtypes between healthy and disease samples,
including cytotoxic markers such as GZMB, GZMK and
PRF1.
Although technologies such as CITE-Seq and AbSeq
allow simultaneous measurements of the surface protein
and gene expression, extracting both the intracellular
protein and gene expression within the same single cell
remains largely unexplored. This is because these
measures require permeabilization of cell membrane
which may result in cell death, thus impairing the
possibility to utilize current approaches for combining
intracellular protein expression quantification with other
modalities, such as scRNA-seq. This roadblock has been
recently addressed by intracellular staining and
sequencing (INs-seq),13 which permits the measurement
of both intracellular protein and mRNA. INs-seq was
applied to several immune subsets, including dendritic
cells, myeloid cells and T cells. For the latter, intracellular
quantification of the transcription factors FOXP3, TCF7
and ID2 in combination with scRNA-seq data revealed
gene modules associated with these transcription factors,
for example, TCF7+ cells had gene modules associated
with na€ıve phenotype (CCR7, SELL and LEF1), whereas
ID2+ cells revealed genes related to cytotoxicity (GNLY,
GZMA/B, PRF1).
Identification of cell state in diseases
Identifying cell states using multimodal analysis can lead
to the discovery of novel correlates of disease, clinical
 Table 1. Overview of the current applications of single-cell multimodal analysis to study immune cells

Applications Targeted
to immunology Genome Epigenome Transcriptome proteins Component details Reference
8
Molecular cell state U U Surface protein (sorting) + mRNA
29
U U Surface protein (sorting) + TF binding + chromatin accessibility + clone (TCR)
10,51
U U Surface protein (barcode) + mRNA
26
U U Surface protein (barcode) + mRNA + clone (BCR and TCR)
13
U U Intracellular protein (sorting) + mRNA
15–19
U mRNA + clone (TCR)
27
U U U Surface protein (sorting) + mRNA + somatic mutations + clone (BCR)
30
U U U Surface protein (sorting) + mRNA + somatic mutations
29
U U Surface protein (sorting) + TF binding + chromatin accessibility + clone (TCR)

5,6
Temporal U U Surface protein (sorting) + mRNA + pseudotime (mRNA)
36
U U Surface protein (sorting) + chromatin accessibility + pseudotime (chromatin accessibility)
35,62
U mRNA + pseudotime (mRNA)
44
U mRNA + pseudotime (mRNA) + clone (TCR)
38
U U Surface protein (sorting) + mRNA + clone (barcode)
63
U U Surface protein (sorting) + mRNA + clone (genetics)
34
U U U Surface protein (sorting) + mRNA + chromatin accessibility + clone (mitochondrial DNA)
39
U U Surface protein (sorting) + chromatin accessibility + clone (mitochondrial DNA)
33
U U Surface protein (sorting) + mRNA + clone (TCR)
37
U U Surface protein (sorting) + mRNA + clone (BCR)
64,65
U U Surface protein (sorting) + mRNA + clone (Ag-specific TCR)

41
Spatial U U Surface protein (sorting) + mRNA + spatial (cell location)
3,4,43,44
U mRNA + spatial (cell–cell)

BCR, B-cell receptor; mRNA, messenger RNA; TCR, T-cell receptor; TF, transcription factor.

33
 parameters and outcome. For example, a study
performed proteomic and transcriptomic analysis using
CITE-Seq and scRNA-seq from the peripheral blood
mononuclear cells of healthy individuals vaccinated with
influenza or yellow fever vaccine.14 This analysis revealed
a distinctive baseline signature across low and high
responders following vaccination. Within each cell type
identified by CITE-Seq protein data, gene expression was
used to identify significant differences between low and
high responders within the plasmacytoid dendritic cell
and lymphocyte clusters, suggesting that people who
respond well to vaccines have a distinct activation status
of cells at baseline (i.e. before vaccination).
Single-cell multimodal analysis has also been utilized to
simultaneously study gene expression and clonal
expansion of T cells and B cells. For instance, gene
expression and immune receptor sequencing from both
of these subsets were simultaneously measured from
peripheral blood mononuclear cells of patients with
metastatic melanoma treated with anti-CTLA-4 and anti-
PD-1 immunocheckpoint blockade.15 By employing
machine learning techniques, the authors of this study
showed that clonally expanded subset of peripheral CD8+
T cells was associated with a long-term treatment
response. Single-cell gene expression and immune
receptor have also been applied to discover new cell states
in cancer such as hepatocellular carcinoma, colorectal
cancer and lung cancer,16–18 as well as in tumor
infiltrating T cells in the context of novel
immunocheckpoint blockade therapies (e.g. in
melanoma).19
In the case of viral infections, single-cell multimodal
analysis has proven extremely useful in the
identification of viral-specific T cells and B cells. These
cells are generally found in low numbers within the
pool of circulating and resident cells, which pose
challenges for their identification and separation for
molecular and phenotypic analyses. Single-cell analysis
has provided a means to accurately characterize rare
cell populations.20 Several teams, including ours, have
applied single-cell multimodal analyses to separate viral-
specific CD8+ T cells using tetramers and then utilized
index sorting and scRNA-seq (Smart-seq2) to
simultaneously identify their gene expression and full-
length TCR in individuals infected with hepatitis C
virus.21,22 These analyses were then used to identify the
active and resting subsets within these viral-specific
responses, along with their clonal expansion. Similar
applications have been also utilized to study chronic
HIV infection, for instance, to demonstrate the
existence of HIV-specific CD8+ T cells that recognize
epitopes within the HLA-II instead of class I23 and
influenza-specific CD8+ T cells,24 to reveal evolving
34
molecular signatures of influenza-specific CD8+ T cells
across different stages of infection.
The importance of single-cell multimodal analysis has
led to several recent studies of coronavirus disease 2019
(COVID-19). Single-cell analysis of both gene expression
profile and immune receptor sequencing has been also
performed on bronchoalveolar lavage fluids from patients
with mild or severe disease.25 This analysis revealed that
patients with mild COVID-19 disease were characterized
by highly clonally expanded CD8+ T cells, and that
proinflammatory monocyte-derived macrophages were
abundant in the bronchoalveolar lavage fluid from severe
COVID-19 cases. The use of proteomics, gene expression
and clonal information has also been investigated.26 Here,
surface protein using CITE-Seq, in addition to scRNA-seq
and B-cell receptor and TCR information, was used to
investigate the peripheral blood mononuclear cell of
COVID-19 patients. These authors showed that a pre-
exhaustion phenotype in HLA-DR+CD38+-activated T
cells and an anti-inflammatory signature in monocytes
are associated with progressive disease, whereas a TCR
and B-cell receptor analysis revealed a skewed clonal
distribution of CD8+ T- and primary B-cell response.
Single-cell multimodal analyses have been recently
applied for the first time in rare pathogenic B cells secreting
autoantibodies in the context of Sj€ogren syndrome.27 In this
study, B cells were first sorted as CD19+CD27+IgD
memory cells from patients with Sj€ ogren syndrome, to
isolate clonally related cells responsible for autoantibodies
associated with cryoglobulinemic vasculitis. By utilizing
single-cell genome and transcriptome sequencing,28 full-
length gene expression data from each cell were analyzed
with VDJPuzzle22 to reconstruct the full-length heavy and
light chains of immunoglobulin B cell secreting
autoantibodies, thus demonstrating the expansion of a
single “rogue” clone dominating the observed phenotype.
Single-cell DNA was then utilized to identify lymphoma
driver somatic mutations present only within the rogue
clone of autoantibody-forming B cells. This study provided
the first direct evidence that somatic mutations drive loss of
tolerance and disease pathogenesis.
Single-cell multimodal analysis has also been useful to
investigate the epigenetic profile of T-cell subsets and
their clonal expansion in the context of leukemia.29 By
combining assay for transposase-accessible chromatin
using sequencing with TCR sequencing, this study first
identified regulatory elements and transcription factors
associated with each canonical T-cell subset in healthy
donors. Surprisingly, this study found that the epigenetic
profiles of canonical T cell subsets form a continuum of
states, suggesting significant regulatory variability within
cell surface marker-defined subpopulations. By applying
this approach to T cells derived from leukemia patients
the authors identified the state of abnormal clones, hence
determining the mechanisms driving disease. In a
separate study,30 mutations from scRNA-seq data were
used to identify and isolate three clones in a bone
marrow sample from a patient with acute myeloid
leukemia. Gene expression was then used to identify the
cell-type compositions of these clones, determining that
these clones belonged to progenitor-like, monocyte-like
and dendritic cell-like cells.
TEMPORAL ANALYSES
As discussed, multimodal measurements of immune cells
can lead to a deeper understanding of the heterogeneity
inherent in these immune cells, and the changes that a
disease can cause to cell state. However, the molecular state
of an immune cell is a dynamic process, from HSC
generation in the bone marrow to its differential fate.
Single-cell measurements are crucial in obtaining an
accurate estimation of this temporal differentiation. This is
because bulk samples contain a mixture of cells at various
differential stages, thus tracking the average bulk expression
across time may not reflect the terminal differential
trajectory.31 In order to study the evolving state along a cell
lineage, the molecular-state modalities will need to be
coupled with temporal modalities. We define temporal
modalities as information related to the time ordering of
cells during their differentiation process. The ideal scenario
would be to obtain measurements of cell states belonging to
the same clone at different time points. However, this
information is not always available, for instance, from
cross-sectional studies. Recent single-cell technologies have
attempted to address these issues which have allowed for
the study of cell state32 and clonal differentiation.33,34
Molecular cell state differentiation
Numerous algorithms have been proposed to estimate
time information for each cell with a metric known as
“pseudotime” using either gene expression or chromatin
accessibility data.32 This metric describes how a modality
changes in a continuous differentiation process along a
trajectory. To obtain this trajectory, a dimension
reduction step is first performed so that each cell is
embedded in a lower dimensional space. A trajectory is
then formed in this space, with cells positioned along this
trajectory depending on their transcriptional or
accessibility profiles.32 Despite pseudotime values being
only an estimate of how cell state evolves over time, and
clonal information is not known for each cell, numerous
immunological discoveries have already been made using
this metric, which we will now review.
HSCs are stem cells derived from the bone marrow
which give rise to myeloid and lymphoid lineages and are
thus a natural starting point to study pseudotime using
single-cell multimodal analysis. Several works have
utilized the natural inherent relationship between HSCs
and differentiated immune cells, to infer the
differentiation trajectories using single-cell genomics. For
instance, differentiation trajectories were obtained from
scRNA-seq data of HSCs from the bone marrow of
mouse,5,6 which revealed three differentiation
trajectories,5 originating from sorted CD48CD150+
CD45+EPCR+ HSCs, and ending with erythroid,
granulocytes–macrophage and lymphoid progenitors.
Another natural avenue for pseudotime analysis is the
study of T-cell selection in the thymus. In a recent
study,35 transcriptomic data were obtained from
developing and postnatal thymus and postnatal samples
covering the entire period of active thymic function.
Pseudotime values obtained from scRNA-seq revealed
developmental marker genes for different cell types
during T-cell development, such as ST18 for early double
negative, and AQP3 for double positive. The TCR was
also obtained, which revealed that the dependence of
nonproductive on productive recombination events was
associated with different cell types. For example, there
was a higher amount of fully recombined TCRb
compared with nonproductive chains in double-negative
stages that dropped to basal levels as cells entered double-
positive stages, thus demonstrating the impact of thymic
selection on the TCR repertoire.
Although pseudotime trajectories have been mostly
derived from scRNA-seq data, this metric can be also
obtained from other modalities, such as single-cell
chromatin accessibility data. For example, in a recent
study, cellular populations were sorted from CD34+
human bone marrow cells, including myeloid, erythroid
and lymphoid lineages.36 Pseudotime was then generated
from chromatin accessibility data, which showed motif
accessibility dynamics along myeloid cell differentiation.
For example, they showed that accessibility at
transcription factor motifs associated with HOXB8 and
GATA1 was high in HSC and decreased through
differentiation to common myeloid progenitors.
Clonal differentiation
Although trajectory analyses using pseudotime have
provided important insights into cell state differentiation,
these approaches have limitations in revealing the true
cell lineage endpoint. To achieve this goal, novel methods
have been developed which can identify clonal markers in
individual cells, in addition to also measuring “omes”
35
 and temporal information in the same single cell. We will
discuss several of these approaches.
The most natural way to track clones in T cells and B
cells is by their unique cell receptor. In the context of
cellular immunotherapies, such as chimeric antigen
receptor (CAR) T cells, single-cell multimodal analysis
have been recently applied to study clonality, gene
signatures and kinetics. TCRs were used to track CAR-T
cells in patients undergoing anti-CD19 CAR-T
immunotherapy in leukemia, in order to understand
characteristics of clonally expanded CAR-T cells.33 In this
study, CAR-T cells were sorted from blood samples from
patients with B-cell acute or chronic lymphoblastic
leukemia to isolate CD8+ CAR-T cells using a truncated
version of the epidermal growth factor receptor, which is
coexpressed with the CAR on the T-cell surface. A
decrease in TCR diversity was observed after CAR-T
infusion, suggesting that CAR-T cells underwent clonal
expansion. Gene expression analysis showed clones which
increase in frequency after infusion displayed higher
expression of cytotoxic genes. Gene expression analysis of
the infusion product showed distinct clusters
distinguished by expression of activation, cytotoxicity,
mitochondrial and cell cycle-associated genes. Tracking
clones via their immune receptor has been also applied to
autoimmune diseases.37 Transitional IgDlow B cells were
first sorted from peripheral blood mononuclear cell
collected longitudinally from patients with myasthenia
gravis who relapsed after treatment with rituximab, a B-
cell-depleting drug. B-cell receptor clones were then
isolated using the gene expression data, which were
shown to be related to clones identified previously from
untreated patients. This then allowed identification of
persistent B cells. Clustering using gene expression
revealed 820 persistent clones in both memory B-cell and
antibody-secreting cell clusters.
A recent approach has been to identify clones with
“barcodes,” which can be identified at the single-cell
level. This approach has been applied to HSC using a
lentiviral delivery system.38 Cells cultured in vitro and
cells transplanted in vivo were collected over several days,
and then sorted to isolate oligopotent and multipotent
progenitor cells using flow cytometric markers. In this
study, the early transcriptional signature of HSC was
linked to the clonal fates via barcoding. This high-
throughput system allowed mapping of more than
300 000 cells and 10 968 distinct clones, and identified
genes correlating with fate, revealing two routes of
monocyte differentiation that give rise to distinct subsets
in immune compartments.
Another promising approach to track clones is the use
of mitochondrial DNA.34 This was performed by applying
single-cell chromatin accessibility assay (assay for
36
transposase-accessible chromatin using sequencing) to
cultured CD34+ HSCs, collected over the course of
20 days. Mitochondrial DNA was extracted, which
incrementally accumulates genetic mutations passed onto
daughter cells, and subsequently used for lineage tracing.
Combining lineage tracing with chromatin profiles
revealed possible fates of HSPCs, in particular
distinguishing bipotent progenitors from those biased in
favor of an erythroid versus monocytic fate. Lineage
tracing using mitochondrial DNA has also been applied
to study acute myeloid leukemia.39 Clones were first
isolated based on mutations in the mitochondrial DNA
from assay for transposase-accessible chromatin using
sequencing data, taken from primary blood samples of a
patient with acute myeloid leukemia. This allowed new
insights into “preleukemic” HSCs, adding to the evidence
that this cell population is heterogeneous with multiple
clones, and that the lineage giving rise to acute myeloid
leukemia is not the lineage with the optimal potential
among pluripotent HSCs.
SPATIAL ANALYSES AND CELL–CELL
COMMUNICATION
Multimodal applications at the single-cell level have also
been applied to study how molecular-state modalities are
affected by spatial modalities, in particular a cell’s spatial
location within a tissue, and its location relative to other
cells. Technologies which measure spatial location are
becoming increasingly available,40,41 and some of these
have been already applied in immunology. For example,
single-cell spatially resolved transcriptomics was applied
to mice bone marrow niches using an improved version
of laser-capture microdissection coupled with
sequencing.41 This allowed the transcriptional profile of
major bone marrow cell types to be determined, and
their spatial location in distinct bone marrow niches.
This analysis also showed that Cxcl12-abundant-reticular
cell subsets differentially localize to sinusoidal and
arteriolar surfaces and act locally as “professional
cytokine-secreting-cells.”
Some studies have utilized single-cell multiomics to
investigate cell–cell interactions, and recently applied to
COVID-19. For example, cell–cell interaction was
estimated using CellPhoneDB,42 which was applied to
scRNA-seq data obtained from nasopharyngeal and
bronchial samples in patients with moderate or critical
disease.43 This analysis revealed a higher number of
epithelium–immune cell interactions in patients with
critical COVID-19, in particular for CD8+ T cells,
nonresident macrophages and monocyte-derived
macrophages, thus likely contributing to clinical
observations of heighted inflammatory tissue damage.
Both spatial and temporal single-cell multimodal analysis
have also been also performed on bronchoalveolar lavage
fluid from mild and critical patients.44 TCR clonal
information, gene expression and pseudotime analysis
revealed that patients with mild COVID-19 were
characterized by fully differentiated resident memory T
cells undergoing active clonal expansion, whereas in
critical COVID-19 patients, these resident memory T cells
fail to differentiate or expand. In the same study, the
authors also applied CellPhoneDB to show differences in
immune cell-type interactions between mild and severe
COVID-19. For example, they showed that interactions
between monocytes/macrophages and neutrophils almost
always involve promigratory interactions in critical
COVID-19, but interleukin signaling in mild COVID-19.
Other non-COVID applications include those which
studied cellular interactions between melanoma and head
and neck cancer cells and various immune cells,
including T cells and macrophages3 and isolated T-cell
subsets from transcriptomics data.4
CONCLUSIONS AND FUTURE DIRECTIONS
Single-cell multimodal technologies have led to exciting
discoveries on the mechanisms underpinning the immune
system. We have highlighted some of these discoveries,
which have provided insight into the molecular state of
immune cells, how these states evolve over time and the
impact of spatial location. These technologies are
becoming increasingly available and easy to apply, as
exemplified by the recent publications in the field of
COVID-19 research in the last few months.25,26,43,44
These technologies can also be used to answer more
general questions, such as quantifying the relationship
between transcripts and proteins, or dissecting the
landscape of post-translational modifications. In this area,
there remains more work to be done, as shown by recent
studies investigating promoter accessibility–gene
expression45 and gene expression–protein46 correlation. In
immunology, there have already been significant advances
in the last decade using single-cell multimodal analysis, as
we have reviewed. However, despite these achievements,
there remains promising avenues to be explored. For
example, it is conceivable that with the development of
new multimodal technologies, further cellular states
which comprise of a combination of modalities across
different “omes” will be discovered. Combined with
spatial modalities, these cellular states may also be
spatially dependent. Other promising avenues will now be
discussed.
Novel multimodal technologies are being proposed
every year, with some yet to be applied to immune cells.
For example, an important and yet unresolved problem is
to identify the target genes which are linked to a
transcription factor, as this can lead to a better
understanding of the molecular network modules that
drive immune-cell lineages and their differentiation. Two
multimodal technologies can potentially address this
issue. The first is thiol(SH)-linked alkylation of the
metabolic sequencing of RNA, which integrates scRNA-
seq with metabolic RNA labeling to provide two
modalities in the transcriptome: total RNA levels and
recently transcribed RNA.47 When combined with
perturbation methods, thiol(SH)-linked alkylation of the
metabolic sequencing of RNA can identify target genes of
transcriptional regulators, as has been shown in cancer
cells.48 Single-nucleus chromatin accessibility and mRNA
expression sequencing can also be used to understand
gene regulation, as this technology provides high-
throughput sequencing of the transcriptome and
chromatin accessibility in the same cell.49 Other advances
can be applied for a different problem of characterizing
molecular cell state, and include those utilizing
transcriptomics (mRNA) as one of its modalities, in
addition to either DNA,50 protein expression,10,51
chromatin accessibility52,53 or DNA methylation.54–57
Lineage tracing of immune cells that do not carry a
natural bar code, such as TCR or B-cell receptor, can also
benefit from recent technologies using synthetic barcodes,
such as CellTagging which uses a lentiviral approach.58
This approach offers advantages over alternate
approaches where gene editing is challenging.58
Despite the rapid increase of single-cell multimodal
approaches, several computational and technical caveats
still need to be addressed for optimal analysis of these
data. For example, the sequencing output maybe too
shallow to identify the immune receptor, and optimal
gene expression requires a deeper coverage to control for
technical noise and drop out of low-expressing genes.59
Similarly, better tools with deeper sequencing are
required for identification of more complex gene
expression quantities, such as isoforms. Multimodal
technologies also carry a substantial level of technical
noise which can blur the true biological variation that
exist,1 for instance, between gene and protein expression,
and are an important area for future work. Finally, a
significant challenge is the development of bioinformatics
tools to permit integration of these data, as recently
reviewed.1
The rapid growth of single-cell multimodal
technologies has also generated debate about the precise
definition of how a “mode” or an “omic” is defined. We
have opted for a broad definition, considering a mode as
any type of information from the same single cell, which
is consistent with a previous definition in a highly cited
review.1 We have thus included temporal and spatial
37
 information as separate modalities, in addition to
different information obtained from the same data set.
Temporal and spatial information has also been
considered by other authors as its own separate omic or
modality.60,61 We envisage that future research in this
field will lead to more advanced approaches and methods
to better quantify the temporal and spatial modalities of
immune cells and converge in adopting a less confusing
language, thus increasing the involvement of
immunologists in the field of single cell.
Single-cell data sets are growing remarkably fast. For
instance, the Human Cell Atlas (https://data.humancellatlas.
org), which comprises already approximately 2.7 million
cells, and 19.68 TB of data across multiple tissues in both
healthy and human diseases. This initiative has already
significantly contributed to immunology by providing novel
data sets across thymus, spleen and other organs, as well as
characterizing novel subsets of lymphocytes and monocytes
through development and into adulthood. A major aim of
data-gathering initiatives such as the Human Cell Atlas is to
increase sample size and permit interrogation of single-cell
multimodal data for more complex questions, such as
identifying the entire cell composition of the human body, or
to predict with machine learning algorithms the clinical
outcome in disease. We envisage that single-cell multimodal
technologies will pervade basic and translational immunology
research and will become a tool to discover mechanisms and
new cell states, as well as to mold novel immune therapies to
effectively target specific molecular pathways in disease and
allow the identification of target cells.
ACKNOWLEDGMENTS
This research was supported by a NHMRC Project grant
(APP1121643 to FL). FL is funded by an NHMRC CDA
fellowship (APP1128416).
AUTHOR CONTRIBUTIONS
Raymond HY Louie: Conceptualization; Investigation;
Writing-original draft; Writing-review & editing. Fabio
Luciani: Conceptualization; Investigation; Supervision;
Writing-review & editing.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
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Genomic Cytometry and New Modalities for Deep
Single-Cell Interrogation
Robert Salomon,1,2* Luciano Martelotto,3 Fatima Valdes-Mora,4,5 David Gallego-Ortega1,4,6
1

Institute for Biomedical Materials and
Devices, The University of Technology
Sydney, Ultimo, New South Wales, 2006,
Australia
2
ACRF Child Cancer Liquid Biopsy Program,
Children’s Cancer Institute. Lowy Cancer
Research Centre, University of New South
Wales (UNSW) Sydney, Randwick, New
South Wales, 2031, Australia
3
Centre for Cancer Research, University of
Melbourne, Parkville, Victoria, Australia
4
St Vincent’s Clinical School, Faculty of
Medicine, University of New South Wales
(UNSW) Sydney, Darlinghurst, New South
Wales, 2010, Australia 
5
Cancer Epigenetic Biology and Therapeutics.
Personalised Medicine Theme. Children’s
Cancer Institute. Lowy Cancer Research
Centre, University of New South Wales
(UNSW) Sydney, Randwick, New South
Wales, 2031, Australia
6
Tumour Development Lab, The Kinghorn
Cancer Centre, Garvan Institute of Medical
Research, Darlinghurst, New South Wales,
2010, Australia
Received 6 November 2019; Revised 28
June 2020; Accepted 7 August 2020
* and diseases.
Correspondence to: Robert Salomon,
Institute for Biomedical Materials and
Devices, The University of Technology
Sydney, Ultimo New South Wales 2006,
Australia Email: rob@rob-salomon.com
or rsalomon@ccia.org.au
Published online 5 September 2020 in
Wiley Online Library
(wileyonlinelibrary.com)
DOI: 10.1002/cyto.a.24209
© 2020 International Society for
Advancement of Cytometry
REVIEW ARTICLE
Abstract
In the past few years, the rapid development of single-cell analysis techniques has
allowed for increasingly in-depth analysis of DNA, RNA, protein, and epigenetic states,
at the level of the individual cell. This unprecedented characterization ability has been
enabled through the combination of cytometry, microfluidics, genomics, and informat-
ics. Although traditionally discrete, when properly integrated, these fields create the
synergistic field of Genomic Cytometry. In this review, we look at the individual
methods that together gave rise to the broad field of Genomic Cytometry. We further
outline the basic concepts that drive the field and provide a framework to understand
this increasingly complex, technology-intensive space. Thus, we introduce Genomic
Cytometry as an emerging field and propose that synergistic rationalization of dispa-
rate modalities of cytometry, microfluidics, genomics, and informatics under one
banner will enable massive leaps forward in the understanding of complex biology.
© 2020 International Society for Advancement of Cytometry
Key terms
genomic cytometry; technology; cytometry; genomicsmicrofluidics; single-cell
THE cell is the basic unit of life and is capable of a vast array of biological complex-
ity. In order to understand how different populations of cells can functionally coexist
to form organs, organisms, and indeed disease, it is critical to profile all aspects of
the individual cells. The capability to perform in-depth single-cell analysis has pro-
vided us with a more complete understanding of disease, development, and normal
function. Moreover, the application of single-cell genomic technologies has already
identified many of the molecular features of cell populations within tissues, organs,
Techniques that together comprise the field of Genomic Cytometry have
already been used to reveal a fundamental aspect of biology. Most notably, that cell
populations are more heterogeneous than ever imagined. Each individual cell is
unique in terms of space (e.g., physical position in tissues and/or organs), time
(e.g., phases of cell cycle, activation or developmental state), and molecular profile.
This uniqueness makes understanding the underlying biology a significant challenge.
While in the past, scientists could interrogate, enumerate, and classify cell types
according to their appearance under the microscope, this analysis is limited in the
number of characteristics that can be simultaneously probed, the rate at which
observations can be made has relied heavily on the individual interpreting the data.
Modern flow cytometry emerged to give an additional level of detail to the classifica-
tion process. By making use of multi-parameter, multi-laser instruments, flow cyto-
metry has redefined cell classification at the molecular level, aided the discovery and
definition of major and minor cell subsets, and has quickly become an essential tool
for dissecting the functional complexity of cell populations. In general, however, it is
primarily used to identify cellular protein expression profiles and despite being able
41
 REVIEW ARTICLE
to process many millions of cells in a rapid manner, it is also
hampered by limited dimensionality and the inherent loss of
anatomical context.
Limits around fluorochrome uniqueness and detector
numbers result in characterization ability topping out around
the 30-parameter mark. In line with advances in fluorescent
cytometry and the development of spectral cytometers (1),
instruments such as the CYTOF (2) have matured and are
now capable of 40+ parameters (3, 4). While there is some
debate around the benefits and trade-offs associated with the
use of mass cytometry (5), the advent of scanning ablation
and ion beam systems (6–8) has helped to bridge the gap
between imaging and flow cytometry. In doing so, they have
provided tools that allow 2D reconstruction of tissue sections
such that anatomical location of protein expression can be
performed down to the micrometer range. A recent study by
Keren et al. has improved this resolution down to 260 nm
(9). These imaging systems, however, tend to be much slower
than traditional cytometry and have their own unique
challenges.
Given that even the most advanced methods in fluores-
cence and mass cytometry are still limited, it is clear that new
methods must emerge to allow deep single-cell characteriza-
tion. In order to be widely applicable in biological studies,
these systems should provide throughputs similar to current
fluorescent flow cytometric techniques while also providing
improved dimensionality (hundreds to thousands of parame-
ters simultaneously). By combining advances in cytometry
with the tools emerging from the field of single-cell genomics,
we are entering a new era of Genomic Cytometry. With the
tools and workflows being created by today’s emerging geno-
mic cytometrists, we now can understand, in a concerted
manner, many aspects of individual cells.
Genomic Cytometry techniques, while focused on the
single cell, allow us to identify and characterize a group of
single cells that share a similar function. The characteristics
able to be probed are no longer limited to protein expression
profiles, but now include aspects such as DNA, RNA, pro-
teins, metabolites, and even epigenetic modifications. This
unprecedented ability to sensitively interrogate large numbers
of individual cells at a reduced cost is accelerating discovery
and challenging existing paradigms in cytometry. Perhaps
more importantly, this technological leap is transforming how
we understand basic and translational biology.
The single-cell multi-omics revolution has fostered a par-
allel development of computational approaches, necessary to
integrate and understand the data generated from single-cell
genomic techniques. These methodologies and approaches
have been described elsewhere (10–14). In this review, we
analyze the factors and motivations that have given rise to the
field of Genomic Cytometry. We also provide an overview of
the tools currently available in this space.
UNRAVELING CELLULAR COMPLEXITY
Cells are complex assemblies of macromolecules and
chemicals that function as a single unit during homeostasis,
42
development, and disease. Currently, there are a multitude of
different tools and methodologies that can be used to charac-
terize a cell. These tools can measure the physical characteris-
tics of a cell (such as size, deformability, electrical impedance,
and density) as well as biochemical aspects such as DNA,
RNA, and protein (concentration, monomer composition,
and chemical status including mutation, acetylation, phos-
phorylation, methylation, etc.). Importantly, emerging tools
are increasingly allowing the simultaneous characterization of
these parameters. This is known as multi-omics.
Although many aspects of a cell are able to be assessed
by traditional cytometry, it has primarily been leveraged to
characterize protein expression profiles at the level of the
individual cell. From the early advent of fluorescence cyto-
metry in the late 1960s (15) and cell sorting in 1965 (16), the
underlying technology has remained relatively static. Instru-
ment manufacturers have added additional laser lines and
increased detector numbers in order to improve multiplexed
single-cell characterization; however, flow cytometry is still
hampered by a lack of spectrally unique fluorochromes.
Recent developments in dye technology, particularly around
tunable polymer-based dyes (17), have allowed flow cyto-
metry assays to reach into the 28 color range (18–22). How-
ever, if we look at the total cellular complexity, it is clear that
even high dimensional fluorescent flow cytometry is incapable
of completely characterizing the full range of cellular identi-
ties and cellular states.
To understand the challenge of fully characterizing a sin-
gle cell, we must look at the complexity within cells (Table 1).
The human genome is composed of 3 billion nitrogenous
bases. These are structurally organized into regions that can
be transcribed to RNA and subsequently translated to protein.
These regions are known as genes. Although there is still con-
jecture around the number of genes (24, 25), studies suggest
that the number of human genes sits somewhere in excess of
19,000 (26–29). Of the estimated 19,000 protein-coding genes,
it is possible to make many different proteins, some authors
suggest as many as 100 different proteins can be made from
Table 1. Potential complexity of the individual human cell
ESTIMATED OBSERVABLE
MEASURABLE CHARACTERISTIC NUMBERS
DNA 3,000,000,000 (bases)
Epigenetic states
Open chromatin regions 100,000–150,000 (peaks)
(enhancers and
promoters)
DNA methylation 25,000 (CpG islands)
Three-dimensional genome 7,000,000 (long-range
architecture contacts) (23)
RNA 19,000 (coding genes)–
100,000 (noncoding
RNAs)
Proteins >19,000
CD markers >400

each gene (30). To date, the Human Cell Differentiation Mol-
ecule (HCDM) group has defined over 400 cluster of differen-
tiation markers (31).
In addition to regions that code for proteins, noncoding
regions of the DNA also exist. These regions include
enhancers, insulators, and promoters, which are key for gene
expression regulation and thus important markers of cell-
type. Epigenetic mechanisms like DNA methylation, histone
post-translational modifications, expression of noncoding
RNAs, three-dimensional, structure and nucleosome position-
ing all shape the conformation of the chromatin to regulate
gene transcription adding an additional layer of complexity to
the characteristics of the cell (32).
COMMON GENOMIC CYTOMETRY APPROACHES
Broadly speaking, it is possible to arrange Genomic Cyto-
metry techniques into five main methodology categories.
These categories are shown in Figure 1, they are:
1. Plate-based approaches (making use of traditional Fluo-
rescent Activated Cell Sorting [FACS]).
2. Microfluidics: (1) Droplet-based microfluidics (aqueous
reaction chambers created within an oil-in-water droplet);
and (2) solid microfluidics (miniaturized single-cell han-
dling tools with associated molecular workflows for
downstream characterization).
3. In situ combinatorial indexing (using the cell as the reac-
tion chamber itself).
4. Image-based approaches (making use of direct imaging
or spatially traceable barcodes to create high dimensional,
anatomically relevant images).
Plate based
In situ Droplet
microfluidic
Genomic
cytometry
methodology
categories
Spatial Solid
microfluidic
Imaging
Figure 1. The main methodology categories that comprise the
field of Genomic Cytometry. [Color figure can be viewed at
wileyonlinelibrary.com]
REVIEW ARTICLE
5. Spatial transcriptomics (combining basic imaging with
novel positionally traceable cellular barcodes).
Plate-Based Approaches
Plate-based assays are the most familiar to the traditional
cytometrist and are one of the few high throughput Genomic
Cytometry methods that currently allow active single-cell
deposition. Active cell deposition is usually achieved using
FACS, which allows selective deposition of cells based on
characteristics measurable by traditional flow cytometry
techniques.
Mechanically, plate sorting is most commonly achieved
through the use of electrostatic droplet-based cell sorting. In
these systems, single cells are sequentially flown through an
interrogation point, characterized and deflected into the well
of a microtiter plate. By incorporating a system capable of
moving the microtiter plate with repeated micron-level accu-
racy, it is possible to target individual wells sequentially. Cells
are deposited into 96- or 384-well microtiter plates; however,
in some cases higher density plates can be used. In addition
to ensuring the target cell is deposited into the correct well,
most instruments will allow the operator to control the likeli-
hood that (1) a cell is in the deflected drop, (2) more than
one target cell is not deposited, and (3) the nontarget cell
contamination is minimized. For traditional FACS, these are
controlled through the application of a sort mask and allow
the operator to balance cellular throughput and deflection
accuracy with the requirements of high-speed cell sorting.
As current cytometers have not yet overcome the
randomness of cell arrival times, many cells that meet the
selection criteria are not deposited into the sort well. Single-
cell masks look at the predicted position of the cell in the
individual drop and will abort the sort if the cell is located in
either the leading or trailing edge of the drop. This means
that single cells on the periphery of the drop are not deflected
and adds to the cell losses associated with the requirement to
abort sort packets that contain coincident events. The ability
to deterministically control cell location with relation to time
and space will remove inefficiencies associated with the Poisson
distribution of cells in drops and will result in higher through-
put, lower loss single-cell approaches, while still retaining the
characterization complexity afforded by traditional FACS.
While electrostatic droplet-based FACS is by far the
most common method for depositing cells into microtiter
plates, emerging technologies such as the CellenONE and the
WOLF cell sorters are providing alternatives. Both of these
systems use a low-pressure microfluidics-based approach and
can thus be used on highly friable cell types that may be sen-
sitive to the stresses of traditional FACS. The CellenONE sys-
tem is a unique ultra-low volume liquid handler that utilizes
an active image-based cell sorting approach to improve cell
deposition accuracy while simultaneously minimizing cell loss
(sort aborts are simply collected without dilution for subse-
quent reanalysis and deposition). Both the WOLF and the
CellenONE systems are slow when compared to FACS, and
can only handle limited cell numbers, for this reason, they
43
 REVIEW ARTICLE
tend to have specific applications and often require pre-
enrichment steps when dealing with rare cell populations.
Modern FACS instruments also include a software mod-
ule that tracks the characteristics of the cell sorted and links
this to the well coordinates. This process, known as index
sorting, is critical to multi-omic studies as it allows protein
expression profiles (captured as part of the sort decision) to
be cross-correlated to the genomic data generated in down-
stream assays.
Assays that take advantage of a plate-based approach
include: Smart-Seq (33), Smart-Seq2 (34), Smart-Seq3 (35),
STRT-seq (36), STRT-seq-2i (37), Cell-Seq, Cell-Seq2 (38),
MARS-Seq (39), mcSCRB-seq (40), Qartz-seq (41), Qartz-
seq2 (42), scBS-seq (43), and single-cell HiC (44).
Microfluidics
Microfluidics have expanded massively in popularity in the
past two decades (45). In recent years, the field has also made
a significant contribution to both our understanding of biol-
ogy and to many areas of health care (45–48). Using micro-
fluidics, entirely new assays can be created and traditional
assays miniaturized. With reactions performed in the nano to
pico-liter range (49), microfluidic-driven miniaturization can
result in log fold difference in the reaction volume. Because
miniaturization can improve reaction efficiencies by simulta-
neously reducing reagent and sample input, microfluidics is
becoming increasingly critical to our ability to perform high-
throughput, high-resolution, high-sensitive assays in a cost-
effective manner.
Microfluidics is used in a range of technologies but with
reference to genomics, its application in massively parallel
sequencing technologies was a significant contributor to the
precipitous drop in sequencing cost. It is also being used in
most of today’s commercially available, high-throughput
Genomic Cytometry platforms, such as the 10× Genomics
Chromium and BD Rhapsody, Dolomite Bio Nadia, Mis-
sionbio Tapestri, ICell8, Biorad ddseq, InDrops, and Fluidgm
C1 systems. To assist with the categorization of the many
microfluidic approaches available, we have split the tech-
niques into two subcategories, those that involve droplets and
those that utilize miniaturized solid reaction chambers.
Droplet Microfluidics
The realization that droplet microfluidics is useful in the
study of biology came of age with the simultaneous publica-
tion of two seminal papers out of Harvard and the Broad
Institutes in 2015 (50, 51). These papers showed, for the first
time, the application of high-throughput droplet-based gener-
ators in single-cell RNA-seq (scRNA-seq). Since then, com-
mercial systems such as the 10x Genomics Chromium, Biorad
ddseq, Dolomite Bio Nadia, and the Missionbio Tapestri sys-
tems have been released. Among these, the 10× Genomics
Chromium system has the broadest acceptance. This is likely
due to the fact that it was the first to include a highly defined
kit-based approach combined with an accessible data inter-
face. At the time, this created a uniquely user-friendly ecosys-
tem. With this, a biologist without deep expertise in
44
microfluidics and genomics could generate single-cell data
with relative ease. As the field becomes more mature and
competitors increasingly enter the market, we expect the
dominance of a single platform to be significantly challenged.
Mechanistically, droplet-based microfluidic systems work
by mixing two immiscible liquids to create a water-in-oil
emulsion. The oil forms a self-contained reaction vessel
around an aqueous phase. The aqueous phase contains both
cells and a bead containing uniquely barcoded mRNA capture
probes in lysis buffer. For 30 scRNA-seq assays, the capture
probe contains a poly dT region of around 22–25 nucleotides,
which binds to polyadenylated transcripts released upon cell
lysis. Thus, as the mRNA is released the polyadenylated
region of the transcript is immediately bound to an oligo con-
taining a (1) a cell barcode, (2) a Unique Molecular Identifier
(UMI), and (3) a nucleotide region that assists with subse-
quent transcript amplification. As the aim of these systems is
to co-locate a single bead with a single cell to a single droplet,
the RNA profile for each captured cell can be obtained by
informatically pooling of cell barcodes. The UMI tracks indi-
vidual transcripts allowing for correction of amplification
bias. The utilization of the dual barcode approach allows digi-
tal transcript counting at the level of the single cell.
Droplet volume is dependent on the flow rates and the
chip geometry and while this can be used to create a wide
range of droplet sizes, the droplets used in scRNA-seq appli-
cations generally range from a few hundred pico-liters to a
few nano-liters (52). Droplet volume has been shown to be
inversely related to the number of transcripts detected in the
final library, for this reason, applications such as DroNC-Seq
(designed for polyadenylated RNA transcripts from the cell
nucleus) are better suited to systems that produce smaller
droplets (75 vs 120 μm diameter droplets) (53).
Droplet microfluidics have been extensively utilized in
the context of Genomic Cytometry. In addition to the work
mentioned above, it has been used to profile transcriptomes
at single-cell resolution (54), and other non–RNA-based
applications. These include, (1) single-cell epigenetic
approaches such as: single-cell ChIP-seq (55, 56), dscATAC-
seq (57, 58) ChIA-Drop (59) single-cell ATAC seq (23); and
(2) single-cell DNA approaches like single-cell gDNA-
seq (60–62) and a variety of multi-omic workflows including
CITE-Seq (63), REAP-seq (64) (protein and transcriptome),
ECCITE-seq (65) (transcriptome, protein, clonotypes, and
CRISPR perturbations), and SNARE-seq (66) (chromatin and
transcriptome).
Solid Microfluidics
Solid microfluidic platforms use physical barriers to create
individual reaction chambers, often at high physical densities
but always with ultra-low volumes. These chambers can be
made from a variety of materials but commonly include plas-
tic, metal or polydimethylsiloxane (PDMS). Because solid
microfluidics uses a physical confinement on solid substrates,
it is possible to perform imaging on the cells in the well. If
each well location can be associated with the unique cell
barcode, then it is also possible to associate this data with the

downstream genomic characterization. Systems that allow this
tend to have lower throughput and include the Fluidgm C1™
and ICell8™ systems.
The Fluidgm C1™ system is perhaps the best know solid
microfluidics platform. The C1 utilizes an intricate micro-
fluidics architecture to provide high-level control of the com-
plex molecular reactions required for single-cell analysis. The
C1 system has been used in a number of studies characteriz-
ing single cells at the level of RNA, DNA, and epigenetic
changes (67–69). Despite the systems advanced approach,
problems have been identified and care should be taken with
its use (70). The ICell8™ system is a commercially miniatur-
ized plate-based system that allows high-density fluid han-
dling to achieve microfluidic scale single-cell genomics.
Recently, Becton Dickinson has released a high through-
put scRNA-seq system, the BD Rhapsody. It uses a similar
approach to the CytoSeq (71) and Seqwell protocols (72). By
using a microwell approach, the Rhapsody system can place a
single bead in virtually every well and does not expose the
cells to the same pressures associated with droplet generation.
This may be an important consideration when working with
cells highly sensitive to pressure-related stress.
In addition to high recovery rates, Rhapsody workflows
also allow both a whole transcriptome as well as a targeted
transcriptomics approach. While commercial modifications
have occurred, the molecular workflow is similar to that used
in many droplet-based scRNA-seq techniques. The targeted
scRNA-seq approach, however, is currently unique to the
Rhapsody and while it requires a priori knowledge of the sys-
tem being interrogated, it allows transcripts of interest to be
deeply probed without incurring the high sequencing cost
associated with reading common housekeeping and lowly
informative transcripts. Depending on the panel, it is possible
to obtain the same sequencing saturation, with up to 10 times
less sequencing reads than that obtained when using a WTA
approach (73).
In Situ Combinatorial Indexing
In situ single-cell methods provide an ingenious way to use
the inherent structure of the cell or nuclei as the reaction
chamber itself. This is achieved by first fixing the cell using
methanol, or beginning with an intact nucleus, and subjecting
these to multiple sequential barcoding steps using a split-pool
approach. Through successive integration of molecular
barcodes into the cell/nucleus itself, in situ combinatorial
methods are capable of building up a library of uniquely
barcoded single cells. For these methods to work effectively, it
is critical to ensure that the number of barcodes that can be
created is well in excess of the number of cells/nuclei being
labeled. As the total number of barcodes possible is a combi-
nation of (1) the number of unique starting oligos and (2) the
number of successive split-barcode-pool-split steps, these
methods require careful balancing of cell inputs to available
barcodes. Failure to do this will result in cells/nuclei sharing
the same barcode.
Notable examples of in situ combinatorial approaches
include, a 2015 method to perform single-cell combinatorial
REVIEW ARTICLE
indexing ATAC seq (74), sci-RNA-seq (single-cell combinato-
rial indexing RNA sequencing) (75), split-pool ligation-based
transcriptome sequencing (SPLiT-seq) (76), single-cell combi-
natorial indexed sequencing (SCI-seq) (77), sci-CAR (78)
single-cell transposome hypersensitive sites sequencing (THS-
seq) (79), single-cell DNA methylation (sci-MET) (80),
droplet-based sci-ATAC (57), and single-cell Hi-C (Sci-Hi-C)
(81). Recently, SplitBio announced commercial release of a
single-cell RNA sequencing kit utilizing in situ combinatorial
indexing.
Image-Based Approaches
In contrast to spatial transcriptomic systems that rely primar-
ily on spatially attributable cell barcodes, image-based Geno-
mic Cytometry techniques rely on in situ imaging of cells.
These systems have been used to directly image the location
of both RNA and protein in tissue sections. Because sample
handling is reduced and solid tissue does not require diges-
tion, such systems may provide the most representative
method to study cellular composition in solid tissues. Exam-
ple systems include the Codex and a number of highly multi-
plexed fluorescent in situ hybridization (FISH)-based
approaches.
Highly multiplexed FISH approaches take advantage of
specially designed probes combined with multiple rounds of
hybridization and imagining to build anatomically localized
transcript maps on tissue sections. Examples of such
approaches include MERFISH (82), STAR-map (83), Seq-Fish
+ (84), or DNA microscopy (84).
The Codex system (85) can perform high-dimensional
image-based protein detection with the use of oligo-
conjugated antibodies. The system has been adapted for both
slide imaging, super-resolution imaging, and has also been
shown to work with volumetric imaging. By using a series of
fluorescently labeled bases and relying on the specificity of
complementary binding of fluorescently labeled base pair
sequence to the oligo attached to the antibody, it is possible
to perform highly multiplexed protein detection in tissue.
Codex has been validated in both FFPE and frozen samples
and can detect more than 40 proteins from the same
individual cell.
Spatial Transcriptomics
Spatial transcriptomic workflows are complicated and require
complex bioinformatics pathways. However, they can be sim-
plified to a number of key steps, (1) a tissue section is cut,
(2) section is laid on a solid imageable surface containing
immobilized region-specific capture probes (these are akin to
the cellular barcode used in other methods), (3) the section is
imaged, (4) the sample is then permeabilized, and finally
(5) the polyadenylated mRNA is captured by spatial probes.
Following this, cDNA is synthesized, libraries are created, and
then sequenced. As the location of the unique oligo sequence
for the capture probe can be traced back to a discrete physical
location, it is possible to create a single-cell transcriptomic
library that retains anatomical information. The resolution of
the system is governed by both the spot size of the deposited
45
 REVIEW ARTICLE
capture probes and the distance between the centers of adja-
cent capture probe spots. The very first spatial transcriptomic
system (86) had a spot size of 100 μm, with a distance
between spot centers of 200 μm, and an estimated 200 million
capture oligos per spot. Academic systems with spot sizes
approaching that of the single-cell include Slide-seq (87) and
HDST (88). These systems have a resolution of 10 and 2 μm,
respectively. Recently, alterations to the molecular compo-
nent, including “the bead barcode synthesis, array sequencing
pipeline and the enzymatic processing of cDNA” of the Slide-
seq method, were used to improve sensitivity by an order of
magnitude (Slide-seqV2) and allow better transcript represen-
tation (89).
Commercial methods such as the Visium from 10×
Genomics are currently available but not yet in widespread
use. These methods are also not yet at the level of the single
cell. Instead, they have spot sizes that contain many cells and
have large gaps between the spots. The Visium platform uses
spot sizes of 55 μm, with the separation between spot centers
being 100 μm. One caveat of systems like this is the need of
permeabilization time optimization which will vary from
sample to sample. We expect that as spatial transcriptomics
are further developed, they will become a valuable method for
deeply characterizing patient disease. However, until stan-
dardized protocols across a number of tissue types can be
determined, the widespread clinic adoption of such systems
will likely be hindered.
MULTI-OMICS
Multi-omics is the science of combining measurements
afforded by the different omics modalities on the same sam-
ple. In Genomic Cytometry, multi-omics involves the mea-
surement of more than one class of cellular characteristics at
the level of the single cell simultaneously. Generally, this
includes the measurement of (1) RNA with protein, (2) RNA
with DNA, (3) DNA with protein, or (4) epigenetics analysis
with protein. However, approaches allowing three modalities
to be probed simultaneously are emerging.
Low throughput multi-omics has been possible since the
advent of FACS-based index sorting for downstream scRNA-
seq applications. By simply varying the downstream genomic
analysis method, it is possible to use index sorting for a multi-
tude of single-cell multi-omic studies. This approach is often
used in mid throughput scRNA-seq plate-based assays such
as Smart-Seq (33, 34), Cell-Seq2 (38), and MARS-seq (39).
Even inherently multi-omics methods such as G&T-seq (90)
can be combined with index sorting to add a protein dimen-
sion to the multi-omic analysis. The idea of using index
sorting to boost multi-omics identification of single-cell at the
level of RNA, DNA, and protein has recently been leveraged
in the TARGET-Seq (91) protocol.
In order to facilitate high-throughput multi-omic approaches
involving protein detection, a number of oligonucleotide-
conjugated antibody techniques have been developed. These
include CITE-seq (63), REAP-seq (64), and Ab-seq (92). The
use of oligonucleotide labeled antibodies has allowed a
46
substantial step forward in the ability to perform high dimen-
sional single-cell protein detection. By incorporating a unique
oligo onto the antibody, it is possible to detect the extra-
cellular protein expression on the cell using common 30
scRNA-seq. The oligos attached to the antibodies contain
(1) an antibody specific base pair sequence (to identify anti-
gen specificity), (2) a PCR handle (to allow amplification dur-
ing library preparation), and (3) a poly-A sequence (to allow
the antibody conjugated oligo to be captured by the polyT
region of the capture probe). This approach has been com-
mercialized by both Biolegend and Becton Dickinson.
The use of oligonucleotide-conjugated antibodies has
been shown to be effective at detecting many antigens. How-
ever, the technology is relatively new, and care should still be
taken when designing panels. While we do not yet have
guidelines for panel design, factors such as (1) epitope expres-
sion density, (2) cell numbers stained, (3) sequencing depth,
(4) relative expression ratios, and (5) library complexity are
likely to affect the outcome of oligo antibody characterization
studies.
One of the criticisms of oligonucleotide-conjugated anti-
bodies is that the sequence allocation required to detect all
bound antibodies is dependent on the relative expression
across all proteins in the panel. In panels that contain a few
very high-expressing antigens, most of the sequence reads can
be taken up by a small number of antigens. In this case, the
dynamic range of the remaining antibodies is significantly
reduced. While there are a number of ways to approach this
(including antibody titration and spiking in cold, unlabeled
antibody), one approach that will undoubtedly become popu-
lar is to first sort populations of cells defined by high
expressing antigens using fluorescently labeled antibodies
prior to labelling sorted fractions with oligo-antibodies to
identify the remaining antigen profiles.
This FACS-assisted sequencing approach ensures an
efficient use of sequencing reads and when combined with
hashtag antibodies (93) or lipid modified oligo or cholesterol
modified oligo (94) (to molecularly barcode each sorted
population), it becomes a powerful multi-omics strategy with
high-throughput. A comparison of this approach, including
its impact on sequencing read allocation, is modeled in
Figure 2.
APPLICATION OF GENOMIC CYTOMETRY
Since around 2015, there has been an explosion of methods
aimed at single-cell genomic characterization. Alongside this,
there have been an increasing number of studies making use
of scRNA-seq approaches; see review (95). Indeed, following
the completion of the human genome project (96), scientists
have become increasingly aware that bulk genomic
approaches lack the precision to unravel subtle changes at the
level of the individual cell. This is critically important in dis-
eases such as cancer and immune disorders where a single
rogue cell can be the base of disease. It is also important for
the understanding of many developmental processes where
single cells give rise to many cells.
 REVIEW ARTICLE

Figure 2. FACS assisted sequencing provides an efficient and targeted multi-omics approach. (A) A comparison of standard full oligo
antibody panel (unbiased) (top), with FACS assisted sequencing (targeted) (bottom) using a combination of fluorescently labeled
antibodies (for pre selection of populations) followed by oligo antibody labelling. (B) Read sequencing utilization in a mock panel. An
imaginary 30 plex panel was created. The panel consisted of 5 high-expression epitopes, 14 medium-density epitopes, and 11 low
expressors. To compare the effect of removing the high-expressing antigens from the sequencing run, we compared the relative
proportion of sequencing reads used by each oligo tag under both conditions. Each of the concentric circles in the radar plots indicates a
single percentage of sequencing reads used up by the marker. This model predicts that when highly expressed antigens were removed
from the oligo antibody panel, it is clear that low-expressing antigens are associated with higher read counts when FACS assisted
sequencing was used. [Color figure can be viewed at wileyonlinelibrary.com]

Whole transcriptome analysis, the method used by the
majority of scRNA-seq studies to date, allows global profiling
of many of the RNA species found in cells in an unbiased
manner and without the need of a priori knowledge of the
cells or the cell system to be studied. Although single-cell
transcriptomic methods are not capable of amplifying every
single mRNA, even relatively poorly performing methods are
proving capable of accurately identifying many existing and
novel cell populations (97). Furthermore, many of the original
methodologies are being improved with molecular techniques
aimed at increasing transcript detection sensitivity. Notable
examples include Chromium V3, Smart-Seq 3, Quartz-Seq2,
and Seq-Well S^3 (35, 42, 98, 99).
Early studies have tended to be descriptive efforts, pri-
marily aimed at uncovering the cellular heterogeneity and
identifying unique cell types. These studies have formed the
basis of the Human Cell Atlas (HCA) project (100). The HCA
is a multicenter, international effort aiming to create a database
of all cell types in the human body using single-cell
approaches. This is an important effort and is the next logical
step following on from the human genome project. Just as the
human genome project provides the reference data that has
allowed deep interrogation of the biology associated with geno-
mic changes, the completion of the HCA should provide the
reference data to allow classification of individual cells from
their unique omic signatures. This is particularly important, as
many of the databases that we are currently using to interpret
single-cell genomic studies are based on bulk genomics.
While there is clear virtue in these types of studies, this
shotgun approach is only designed to provide a fundamental

47
 REVIEW ARTICLE
Figure 3. Flowchart for determining the most suitable Genomic Cytometry method for the biological question. [Color figure can be
viewed at wileyonlinelibrary.com]
base for more nuanced approaches. The approach required for
biologically directed studies will depend on the (1) biology of
the system, (2) the questions being asked, (3) the technical
expertise of the scientists running the experiment, and (4) the
funds available. While the decision of which technology is best
suited to the biological question being asked is not always
straightforward, we have outlined some of the more common
questions involved in the decision-making process in Figure 3.
CONCLUSION
As we move into the age of Genomic Cytometry, we are now
looking to synergistically leverage the modalities of genomics,
informatics, microfluidics, and cytometry toward a single aim.
To do this, we must develop ways to work in a cross disci-
plinary fashion such that microfluidics and FACS-based tech-
niques can be seamlessly integrated into molecular workflows
and high-dimensional data analysis frameworks. The combi-
nation of these four, traditionally distinct expertise areas, is
what provides the foundation for the new field of Genomic
Cytometry.
With Genomic Cytometry, it is possible to study cellular
characteristics more deeply than ever before. The new tools
48
emerging to allow RNA-seq, DNA-seq, epigenetic analysis,
and protein detection at the level of the single cell will funda-
mentally change what we know about biological processes
and how quickly we can deeply interrogate complex biological
systems. We are beginning to see a systems-based approach
that will allow us to do accurate single-cell multi-omics stud-
ies with the sensitivity, efficiency, and cost that means true
biology can be uncovered. This deep characterization is all-
owing us to unravel cellular complexity in highly heteroge-
neous samples and to find the root cause of disease and
unravel the cellular complexity of development. Eventually,
we believe it will give us the power to analyze the DNA,
RNA, protein, and epigenetic states of individual cells at
throughputs that will rival that of current flow cytometers.
As the field of single-cell genomics matures, and we
begin to embrace the broader field of Genomic Cytometry, it
will become increasingly more evident that results from
single-cell omics studies will need to be supported and vali-
dated by alternate systems. These systems will include tradi-
tional imaging, lineage tracing, and fluorescence cytometry
methods. This will create a circle of discovery and validation
that unites the field of genomics and cytometry. For this rea-
son, although we envision a dramatic shift in the tools

available to the traditional cytometrist, cytometry will still
hold a critical place in the emerging application of single-cell
genomics. It is, for this reason, Genomic Cytometry will
become the modality of choice for single-cell analysis.
CONFLICT OF INTEREST
The authors declared no potential conflict of interest.
AUTHOR CONTRIBUTIONS
Luciano Martelotto: Conceptualization; writing-review and
editing. Fatima Valdes-Mora: Conceptualization; writing-
review and editing. David Gallego-Ortega: Conceptualiza-
tion; supervision; writing-original draft; writing-review and
editing.
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REVIEW ARTICLE

Computational approaches for high-throughput single-cell

data analysis
Helena Todorov1,2,3 and Yvan Saeys1,2
1 Data Mining and Modelling for Biomedicine, VIB Center for Inflammation Research, Ghent, Belgium
2 Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Belgium
3 Centre International de Recherche en Infectiologie, Inserm, U1111, Universite 
 Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale
rieure de Lyon, Univ Lyon, France
Supe

Keywords
bioinformatics; computational tools;
proteome; single cell; transcriptome
Correspondence
Y. Saeys, Department of Applied
Mathematics, Computer Science and
Statistics, Ghent University,
Technologiepark 927, 9052 Gent, Belgium
Fax: +32 9 221 76 73
Tel: +32 9 331 37 40
E-mail: yvan.saeys@ugent.be
(Received 22 February 2018, revised 4 June
2018, accepted 25 July 2018)
During the past decade, the number of novel technologies to interrogate
biological systems at the single-cell level has skyrocketed. Numerous
approaches for measuring the proteome, genome, transcriptome and epi-
genome at the single-cell level have been pioneered, using a variety of tech-
nologies. All these methods have one thing in common: they generate large
and high-dimensional datasets that require advanced computational mod-
elling tools to highlight and interpret interesting patterns in these data,
potentially leading to novel biological insights and hypotheses. In this
work, we provide an overview of the computational approaches used to
interpret various types of single-cell data in an automated and unbiased
way.

doi:10.1111/febs.14613

Introduction
Single-cell technologies are currently revolutionising
the way life scientists are studying biological systems
from different perspectives. Three major classes of
technologies can be distinguished: imaging-based tech-
niques, techniques based on flow or mass cytometry
and techniques based on next-generation sequencing.
However, this is only a rough classification, as some
recent innovations combine elements of different
classes of techniques. While many of the early data
preprocessing steps are specific to each class of tech-
niques, several downstream computational analyses are
generally applicable to any form of single-cell data,
and one of the goals of this work is to provide a unify-
ing overview of these generally applicable approaches.
Historically, microscopy-based techniques were the
first methodology to study organisms at single-cell
resolution [1]. While initially consisting largely of man-
ual labour and thus being very low-throughput, auto-
mated image acquisition and segmentation have enabled
high-throughput image-based screening, by analysing
up to hundreds of thousands of cells in single-well plates
[2]. Similarly, many other microscopy-based techniques
allow the extraction of information at the single-cell
level, although at a lower throughput. These include
most types of light and electron microscopy, with a
broad variety of applications. Common to all these
image-based approaches is the fact that advanced
image-analysis pipelines are needed to arrive at single-
cell resolution [3]. A typical image processing pipeline
first performs segmentation of the single cells from the
image, followed by a feature extraction step, typically
extracting several hundreds of features for each

Abbreviations
DE, differential expression; HVGs, highly variable genes; scRNA-Seq, single-cell RNA sequencing; TI, trajectory inference.

51
 Computational tools for single-cell data analysis
individual cell [4]. In comparison to other single-cell
approaches where cells are dissociated in suspension, a
major advantage of image-based single-cell profiling
methodology is that it inherently provides the user with
two- or three-dimensional spatial information, as know-
ing a cell’s spatial context is often the key to discover
novel biological findings.
Flow cytometry allows profiling and analysing cells
in a high-throughput fashion and is based on passing
cells through a laser beam in a rapidly flowing fluid
stream. This core technology is in essence very similar
to the original design from the late 1960s [5], illustrat-
ing the robustness of the technology [4,6]. The field of
flow cytometry has emerged as a powerful methodol-
ogy for single-cell analysis due to continuous innova-
tions such as (a) multicolour assays enabling the
measurement of a large number of proteins simultane-
ously [7], (b) spectral flow cytometry [8] in which clas-
sical mirrors, optics and detectors are replaced by
dispersive optics and a linear array of detectors allow-
ing highly complex fluorochrome combinations, (c)
imaging flow cytometry [9] combining flow cytometry
and microscopy for high-throughput imaging of single
cells, and (d) acoustic-based focusing and sorting [10].
In addition, other technological advances such as mass
cytometry have replaced the fluorescent labelling and
readout using optics by labelling using heavy isotopes,
and subsequent readout by mass spectrometry [11].
This eliminates the problem of spectral overlap in clas-
sical flow cytometry, allowing the theoretical measure-
ment of up to 100 proteins simultaneously. Mass
cytometry can also be performed on tissue slices,
thereby scanning the tissue spot-by-spot and perform-
ing a single experiment per spot. This approach,
named imaging mass cytometry, allows performing
spatial proteomics in a high-throughput fashion [12].
The ability to measure increasing amounts of proteins
simultaneously [7] complicates the analysis of this type
of data, which can no longer be analysed manually as
was done with datasets containing a few markers per
cell, but needs new computational approaches to cor-
rectly identify cell populations [13].
Recent developments in microvolume sequencing
have led to a new wave of single-cell ‘-omics’ profiling
technologies [14–18], permitting the quantification of
whole genomes, epigenomes and transcriptomes at the
single-cell level. Novel computational tools are being
developed in order to deal with the continuously
increasing dimensionality of these datasets, since a sin-
gle experiment can quantify molecular characteristics
of up to tens of thousands of cells, measuring tens of
thousands of parameters (e.g. transcripts in the case of
single-cell transcriptomics). A high level of resolution
52
H. Todorov and Y. Saeys
is provided by single-cell omics tools, as they aim to
sequence all of the cell’s content, instead of focusing
on a set of user-defined targets as is done in cytome-
try. This allows performing novel types of analyses,
such as studying the heterogeneity of cell populations
in much greater detail, identifying rare cell types, and
studying the dynamics of cellular systems. Further-
more, the field continues to evolve by combining sin-
gle-cell RNA sequencing with other technologies such
as spatial transcriptomics [19] and CRISPR-mediated
knockout screens (Perturb-Seq [20]/CRISP-seq [21]).
Recent approaches combine transcriptomics with other
types of omics data at a single-cell resolution such as
single-cell proteomics (CITE-seq [22]/REAP-seq [23]),
single-cell genomics (G&T-seq [24]) and single-cell
methylomics (scM&T-seq [25]). These emerging ‘single-
cell multi-omics’ technologies [26] integrate several
types of measurements on the same single cell and are
likely to be part of the everyday methodology of
molecular biologists in the future.
While all techniques described above provide the
user with information at single-cell level, the through-
put, resolution, cost and type of information acquired
differ drastically between technologies. We will take a
computational perspective here, and compare the main
dataset characteristics for the three major classes of
single-cell data introduced above. Classical imaging-
based techniques typically offer a low throughput,
measuring a few hundreds of cells, while more
advanced high-content screening methods allow high-
throughput measurements of hundreds of thousands to
millions of cells. When applying segmentation and fea-
ture extraction, for example using popular pipelines
such as CELLPROFILER [27], almost a thousand image-
derived features can be extracted per cell. However,
many of those capture redundant information and
thus are very correlated. Flow and mass cytometry
allow measuring cells at high throughput, up to mil-
lions of cells for classical flow cytometry. Only a few
tens of parameters can be quantified simultaneously
per single cell, but these parameters often represent
very complementary information, as they are manually
chosen by an expert. Single-cell omics technologies
offer medium throughput, measuring thousands to tens
of thousands of cells in a single run. However, these
data are very rich in information, measuring thousands
of transcripts in the case of single-cell transcriptomics.
While the profiling methodology and dataset charac-
teristics in each of these technologies are very different,
many of the applications and computational workflows
are quite similar. In the remainder of the paper, we
will discuss the differences and commonalities in com-
putational workflows for the different applications.
Computational workflow for
single-cell experiments
Regardless of the specific technology used to generate
a single-cell dataset, a common pipeline can be
devised, starting with the experimental design, data
generation, technology-specific preprocessing, quality
control and subsequent data analysis (Fig. 1). A
detailed design of the experiment is a crucial step
towards minimising technical variation and improving
scientific reproducibility. This not only includes stan-
dardisation of experimental protocols and equipment,
but also careful planning and consultation with statis-
ticians and/or bioinformaticians regarding sample size,
specific setup related to the biological questions that
should be answered or specific types of computational
analyses that should be carried out. Subsequently the
experiment should be performed, ensuring that stan-
dardised procedures are followed for sample prepara-
tion, handling equipment and data acquisition while
appropriate controls are added at multiple steps of the
experiments.
The next step in the pipeline is the preprocessing
and quality control. This step will likely take a consid-
erable amount of time, as it is crucial to start from
good quality data if good quality results are desired.
Therefore, it is important to perform technology-speci-
fic preprocessing steps, a topic that will be covered in
Fig. 1. The computational workflow for single-cell experiments detailed in steps.
the section ‘Data preprocessing and quality control’.
After data preprocessing, an initial exploration of the
data can be performed using visualisation techniques,
in order to perform early detection of any possible
batch effects or unexpected subpopulations. Applying
visualisation techniques may also help to visualise the
population structure within samples, and to compare
this structure between different samples. In this step,
interesting populations or trends may be observed that
require further investigation.
Next, several types of in-depth analyses can be per-
formed, in most cases starting with an automated clus-
tering of the cells into cell types. This clustering allows
quantifying and comparing different cell types in the
samples and identifying new cell types or transition
states. Novel computational approaches to model
gradual transitions between cell states (trajectory infer-
ence) can also be applied at this stage. Other alterna-
tives include specific predictive modelling approaches
such as classification, regression and survival analysis
modelling. All these approaches have the potential to
extract novel biomarkers from single-cell data, with
important diagnostic and therapeutic potential.
Finally, more advanced computational approaches can
be applied to single-cell omics data. The correlations
in gene expression within cells can be studied to assess
gene regulatory networks (network inference). In the
case of multi-omics datasets, data integration
53
 approaches can be used to combine the information
on single-cell mechanisms.
Data preprocessing and quality
control
Single-cell imaging
The preprocessing of single-cell imaging data usually
starts by accounting for batch effects through illumina-
tion correction, and image-wise processing such as
noise removal, aligning or cropping [28,29]. This pro-
cedure is commonly followed by the segmentation of
the individual cells within the images, and finally by a
feature extraction process that yields a vector of
numeric features for each individual cell, usually in a
tabular format.
CELLPROFILER [27] is widely used to extract numerical
features from two-dimensional microscopy images
(such as in high-content screening assays). The main
difficulty faced by CELLPROFILER is the segmentation of
the cells or objects of interest present in the image.
CELLPROFILER contains several fast algorithms that can
extract well-separated objects; however, in many cases,
these objects appear clumped, hindering their segmen-
tation and making it prone to both false negatives
(when the borders between objects cannot be found)
and false positives (when the sensitivity of the detec-
tion is too high). In order to deal with this difficulty,
CELLPROFILER also provides a more complex segmenta-
tion algorithm that follows a hierarchical process: first,
it finds primary level objects that are typically well-
separated (such as cell nuclei, visible on DNA-stain
channels); then, the boundaries of secondary level
objects (such as cell edges) are searched around the
primary level objects.
However, it is also possible that the primary level
objects appear clumped, which is why CELLPROFILER
divides their detection into several steps following the
guidelines of previously published algorithms [30–34].
Clumped objects are first detected, segmented and sep-
arated by dividing lines, thus avoiding false negatives.
Finally, some of the objects are either removed or
merged to reduce the false positive rate. Once the pri-
mary level objects are properly detected, it becomes
simpler to find secondary level objects around them.
CELLPROFILER provides an improved algorithm to prop-
erly detect the borders even when the objects are
clumped against each other. Once the objects have
been segmented, multiple features can be extracted
from each of them in a per-channel basis (area, shape,
intensity, texture, etc.) or at the whole-image level
(number of cells, background intensity, etc.).
54
CELLPROFILER has a modular structure that allows the
user to select and configure the individual algorithms
that will be applied, which in turn defines the specific
preprocessing applied and the features that are
obtained at the end of the pipeline. The resulting fea-
tures can later be used for visualisation, clustering or
differential downstream analyses for instance.
Flow/mass cytometry
In conventional flow cytometry, the first preprocessing
step is typically compensation of the spectral overlap,
to correct for spillover of the fluorescent signal into
neighbouring channels. This is typically accounted for
in the experimental procedure, by measuring the fluo-
rescence of single stains in the different channels,
allowing for the calculation of a compensation matrix.
In mass cytometry, this issue is largely avoided by
using rare isotopes instead of light measurements,
although the measurement of certain isotopes can still
be polluted due to metal impurity levels, oxidation and
abundance sensitivity [35]. Mass cytometry panels
should therefore be designed with caution by pairing
strong intensity markers with less sensitive channels in
order to avoid interference between channels [36]. The
data is then transformed through a biexponential or
hyperbolic arcsine transformation, which improves the
separation between negative and positive cells for the
different markers. Fluctuations in measurements can
also be caused by an unsteady flow rate. Typically, up
to 10 000 cells are measured per second at a steady
rate in flow cytometry. Mass cytometry has a slightly
lower throughput, measuring a few thousand cells per
second. However, obstructions in the fluid stream and
manual interventions can disturb the flow, which also
impacts the amount of protein levels measured. To
remove these technical artefacts, the data needs to be
either manually gated against time or screened by tools
such as FLOWCLEAN [37], FLOWQ [38] and FLOWAI [39],
which can automatically identify and remove sections
in which the flow was perturbed.
The acquisition level of cytometers can slightly
change from one day to another, or even within hours.
The use of control tubes to calibrate the machine
before running an experiment can help to make differ-
ent samples more comparable, but batch effects are
often observed between two experiments. The resulting
slight shift in protein expression can be accounted for
manually, by shifting the gates of every sample that
differs, or in an automated way using the FLOWSTATS
[40] package. In mass cytometry, beads are commonly
used in the experiments, allowing normalisation of the
data based on the signal of these beads to have more
comparable samples. Some markers can also be used
to barcode cells, and then pool several samples
together, to avoid technical bias between different
experimental conditions. When performing experi-
ments on different days, it may be advisable to include
additional control samples, such as an aliquot from
the same sample that is taken along all different exper-
iment days, in order to allow normalisation between
experiment days later on. Once batch effects have been
accounted for, debris, doublets and other low quality
cells can be removed either by manual gating or using
OPENCYTO [41], or FLOWDENSITY [42].
As flow cytometry allows the measurement of pro-
teins at the single-cell level while preserving the integ-
rity of the cells, it is sometimes used to sort specific
cells into wells before sequencing their transcriptome.
The cells can either be sorted by cell population, based
on a set of common markers, or index-sorted, in which
case single cells are sorted into wells and barcoded, so
that their protein expression profile is kept. In this
case, doublets and empty wells might occur, which
should be carefully removed from the analysis before
any further processing step.
Single-cell omics
Preprocessing single-cell omics data based on NGS
technologies further builds on the wide availability of
NGS preprocessing tools that are already available
from experiments on bulk RNA or DNA. However,
single-cell omics technologies lead to a number of
additional challenges when going through the process
from the individual reads to the mapped genomes or
transcriptomes. We will focus here more specifically on
methods for single-cell transcriptomics, as this is the
most widely used type of single-cell omics data at pre-
sent. Several scRNA-Seq protocols were developed,
usually focusing either on sequencing a large number
of cells, or a high amount of genes at an increased
sequencing depth [43]. Due to the low amount of tran-
scripts in the cells, scRNA-Seq data usually contain a
lot of technical variance, requiring specific computa-
tional tools to perform quality control, normalisation
and downstream analyses [44–47].
When performing a computational analysis on
scRNA-Seq data coming from multiple experiments,
batch effects can arise, leading to an increased interex-
perimental variability. Two recently published algo-
rithms can be used in order to reduce batch effects.
These algorithms either identify a gene correlation
structure [48], or a subset of cells coming from the
same population [49], that are shared between the
datasets coming from different experiments. Proper
data transformation is then applied to align similar cell
populations, resulting in more consistent datasets that
can be further analysed together.
Several quality control metrics, such as the library
size and the percentage of mitochondrial genes, are
used to filter out abnormal cells, in order to reduce the
technical variance of the data [50]. Additionally, a
great part of intercellular variability can be caused by
the cell cycle, and it is up to the user to decide whether
this variability should be removed from the data or
not. Cyclone [51] is a method that can be used to pre-
dict the cell cycle stage, which can subsequently be
used to either remove cycling cells, or tag them so that
they can be easily identified later in the analysis. F-
scLVM [52] is another algorithm that identifies the
amount of variability across the expression of each
gene that is due to cell cycle differences. It can be used
to infer ‘corrected’ gene expression values, removing
the effect of the cell cycle.
The next step in the process regards the normalisa-
tion of the count data, since a large part of the
observed variability can be due to differences in size,
viability, capturing efficiency and amplification biases
between cells. Some methods aim to standardise the
total number of reads per cell (RPKM [53], TPM [54],
downsampling) or proportions of the total number of
reads per cell (UQ, full quantile [55]). However, these
methods can be seriously impacted by false negative
counts [56]. Indeed, the number of transcripts in a cell
being very low for certain genes, there is a high proba-
bility that these transcripts will be missed, resulting in
a zero count in the final expression data. These missed
transcripts are called dropouts, and lead to a high
technical variance that can affect the final results.
High-throughput scRNA-Seq protocols typically show
higher dropout rates [43], but high amounts of
sequenced cells can help to infer dropout probabilities.
ZIFA [57] is a method which identifies zero counts
that are most likely resulting from dropout events, and
gives less weight to these counts. ZINB-WAVE [58] is
another method which not only assesses the probabil-
ity for a zero to be a dropout based on the sequencing
depth, but also accounts for batch effects between
samples, and computes global-scaling normalisation
factors, which allow it to be used directly on non-
normalised data.
Some methods rely on spike-ins to distinguish techni-
cal variability from biologically relevant changes in
gene expression [59] (BASICS [60], GRM [61], SAMSTRT
[62]). Spike-ins are control RNA transcripts which are
added in the same quantity to all the samples to be
sequenced. They can be used to normalise the data,
as all cells should have exactly the same amount of
55
 spike-ins after sequencing, and the differences in spike- Table 1. Dimensionality reduction based- and clustering
in amounts should only be the consequence of technical based-tools for visualisation of single-cell high-dimensional data.
artefacts. However, the most commonly used spike-in Class of
set (ERCC [63]) cannot always faithfully account for method Name Description
the intrinsic gene variability, as they have been shown
Dimensionality PCA Linear reduction in the dimensions
to have a length and GC content that differ from mam-
reduction holding the highest variance into
malian transcripts [58]. Moreover, choosing the quan- orthogonal principal components
tity of spike-ins that should be added to the cells can MDS Nonlinear reduction in the
be challenging, as a significant amount of spike-ins has dimensions by preserving the
to be used in order to reflect faithfully the intercellular intercellular distances of high
variability, but may eclipse the intracellular transcripts dimensions in the lower
of interest. However, ERCC spike-ins are still com- dimensions
tSNE Nonlinear dimensionality reduction,
monly used to filter out low quality cells [50]. Overall,
preserves the local similarities
the views on the use of spike-ins for single-cell RNA between cells
Seq normalisation are still conflicting [64–66]. Diffusion Nonlinear dimensionality reduction,
The methods cited above apply global scaling fac- maps computes transition probabilities
tors to all cells equally, assuming that the relation between cells
between the number of genes measured per cell and SPRING k-Nearest Neighbour force directed
graph, preserves the high-
the sequencing depth is the same for all genes. How-
dimensional relationships between
ever, this assumption of a constant gene-count/sequen-
cells
cing depth ratio has been shown to hold on bulk Clustering SPADE Hierarchical clustering of the cells
RNA data, but not in single-cell datasets [67]. Apply- followed by the representation of
ing global scaling factors to scRNA-Seq data might these clusters in a minimal
therefore lead to biased correction of lowly and highly spanning tree
expressed genes. Two algorithms can be used to per- FLOWSOM SOM clustering followed by the
representation of these clusters in
form single-cell specific normalisation of scRNA-Seq
a minimal spanning tree
datasets. The SCnorm method [67] relies on the fact
Scaffold Semisupervised method: new cells
that the normalisation should not be applied in the Maps are grouped with the user-provided
same way to all the genes, as they differ in various cell populations to which they are
properties such as transcript length and GC content. most similar
SCnorm first groups genes with similar dependencies FLOWMAP Hierarchical clustering of the cells,
on sequencing depth and subsequently estimates differ- followed by the representation of
these clusters in a strong
ent scale factors for each group of genes. Alternatively,
connected graph structure
SCRAN [50], first groups cells with similar expression
Phenograph Groups cells which share the same
profiles together, and applies intragroup normalisation neighbours together and identifies
before performing intergroup normalisation. communities which maximise the
Louvain modularity

Visualising high-dimensional
single-cell data
Once the data has been preprocessed, visualisation
tools can help to get a first insight into the structure
of the data. A quick principal component analysis
(PCA) plot of the data can, for instance, allow identi-
fying any remaining source of technical variability
between samples, which should be removed by normal-
isation. Structures in the data or biological differences
between the samples may then be investigated using
different approaches: dimensionality reduction tech-
niques, clustering techniques, or the novel class of
techniques to model cell trajectories and state transi-
tions. A list of visualisation tools and their principal
characteristics is provided in Table 1.
Dimensionality reduction tools aim to capture the
structure of the high-dimensional data by projecting it
to a lower dimensional space that keeps the most
important structural properties of the original, high-
dimensional space. The lower dimensional projection
allows the human expert to visualise and explore the
data. Dimensionality reduction can be performed
either in a linear way (the lower dimensional projec-
tions are a linear combination of the original dimen-
sions), or in a nonlinear way. PCA is a linear
dimensionality reduction technique, in which the fea-
tures with the largest variability are preserved in prin-
cipal components. The main sources of variability in
the data can then be optimally laid out. A PCA can

56
therefore be applied to check for batch effects in the
data, or to identify any main source of variability. The
use of nonlinear dimensionality reduction methods
(e.g. tSNE [t-stochastic neighbour embedding, 68],
MDS [multidimensional scaling, 69], diffusion maps
[70], SPRING [71]) allows optimal plotting of the data
in two dimensions while preserving the local similari-
ties between cells.
Clustering-based visualisation methods group similar
cells together and may be combined with a subsequent
visualisation step, for example by laying out the result-
ing clusters in two dimensions. This reduces computa-
tion time and can simplify the understanding of the
resulting plot. Several methods have been proposed for
the visualisation of clusters in single-cell data (SPADE
[Spanning-tree Progression Analysis of Density-
normalized Events] [72], FLOWSOM [73], FLOWMAP [74]).
These methods represent the clusters under the form
of a graph in which the most similar clusters are linked
by an edge. FLOWSOM also allows performing meta-
clustering, grouping clusters into larger populations,
which has shown to return results very similar to man-
ual labelling of cytometry data [75]. Single-Cell Analy-
sis by Fixed Force- and Landmark-Directed (Scaffold)
maps [76] were specifically designed to simplify the
identification of user defined cell populations in cytom-
etry data. Finally, Phenograph [77] identifies closely
linked communities of cells in a graph structure. This
algorithm therefore identifies populations without any
previous knowledge on the number of expected popu-
lations, which can be very useful in discovery studies.
While most of these methods were initially developed
for flow cytometry data, FlowSOM and Phenograph
are scalable to high dimensional datasets. These meth-
ods can therefore be applied to mass cytometry and
scRNA-Seq datasets, or to features extracted from
images, allowing the visualisation of structure in the
data.
However, scRNA-Seq and image derived data typi-
cally contain much more dimensions than the usual
10–30 colour panels used in cytometry. When dealing
with features extracted from images, a first step can
consist in performing principal component analysis,
which will help to reduce the redundancy of these
highly correlated features. One can then choose to
work with the principal components containing 95%
of the data variability. These principal components
can be analysed as new features, using visualisation or
clustering techniques. scRNA-Seq datasets tend to
contain noise which might bias clustering studies, espe-
cially due to the high amount of lowly expressed genes
and dropouts. Therefore, the highly variable genes
(HVGs) can first be filtered on this type of data
[50,78], which considerably reduces the number of fea-
tures and the noise they contain, while preserving the
main biologically relevant sources of variability.
Another algorithm was implemented in the SEURAT R
package [79] to filter HVGs. Visualisation, clustering
or any downstream analysis algorithms can then be
applied either to the HVGs, or, if the dimensions of
the data are still too high, on the principal compo-
nents of a PCA run on these HVGs.
In order to highlight the differences between the dif-
ferent methods cited above, we applied two dimension-
ality reduction tools (PCA and tSNE) and two
clustering-based tool (FLOWSOM, Phenograph) on a
publicly available scRNA-Seq dataset [16] of 3000
peripheral blood mononuclear cells (PBMCs) from the
10X Genomics platform (Fig. 2). We first preprocessed
the dataset as described in the data preprocessing sec-
tion by filtering out low quality cells and genes. We
then selected the most highly variable genes, to which
we applied the different visualisation methods. This fil-
tering on highly variable genes has two advantages. It
significantly reduces the size of the dataset, therefore
reducing the analysis time, and it helps to focus on the
genes that are driving heterogeneity across cells [50].
The PBMC dataset had previously been expert-labelled
in the Seurat R pipeline [79], which allowed us to use
the cell identities to simplify the comparison of the
outputs from the different methods. The different
methods provided complementary information on the
structure of the data. For instance, all methods except
PCA identified the rare megakaryocyte cell population,
and all methods except FlowSOM represented these
megakaryocyes close to the monocyte cell population.
As a general guideline, it is often advisable to apply
several techniques in parallel to acquire a deeper
understanding of the data structure.
Cell type identification
While the clustering approach to single-cell analysis
assumes that cells are forming well separated groups,
other types of techniques focus on better detecting
cells that are in transition between cell states. In the
first case, the expression of certain markers is expected
to differ drastically, providing hard separations
between cell populations. In the second case, the mark-
ers are seen as continuous variables which smoothly
change from one cell to another, leading to structural
patterns in the data which can be seen as developmen-
tal trajectories (Fig. 3). The choice between the two
sets of methods depends on the biological question,
but a good practice can be to first apply a clustering
algorithm to identify the main populations in the data,
57
 Fig. 2. Comparison of (A) tSNE, (B) PCA, (C) FLOWSOM and (D) Phenograph on the PBMC dataset. (A) The cell colours correspond to the
labels provided by experts in the Seurat R pipeline. (B) The main differences between cell types can be seen on the horizontal (1st principal
component) and vertical (2nd principal component) axis. (C) The colours inside the pies correspond to the cell colours on the tSNE plot. The
background colours correspond to the meta-clusters identified by FlowSOM. Discrepancies between the pie colour and the background
colour highlight the cells for which FlowSOM’s results diverged from the manual annotation. (D) The similarities between the different cell
types are nicely laid out on a Phenograph plot.

and then perform trajectory inference on a specific
group of similar cells. Indeed, trajectory inference tools
will tend to identify trajectories in any dataset, so they
should be applied to specifically delineated sets of cells.
The identification of trajectories in highly variable
datasets is a current challenge, which is only described
recently in the literature [80].
Clustering-based approaches
Several tools have been implemented in order to iden-
tify similar groups of cells in cytometry data, compar-
ing either the similarities between cells (SPADE [81],
FLOWSOM [73]), the distances between cells in a lower
dimensional space (Accense [82]) or the shared neigh-
bours in a graph (Phenograph [77]). A benchmark
study of clustering tools, the FLOWCAP I [83] challenge,
provided several mammalian datasets to assess the
ability of different clustering methods to identify cell
populations accurately. Most tools provided a good
delineation of cell populations compared to manual
gating, and ensemble methods which merged the out-
puts of several clustering methods showed the best
results. However, due to the increasing number of
markers used in cytometry data, there is a need to per-
form benchmark studies regularly, as tools which were
very efficient with low-dimensional datasets might not
necessarily perform equally well in higher dimensions
[84]. Another study [75] compared 18 clustering meth-
ods for conventional flow and mass cytometry data,
taking into account the clustering accuracy as well as
the computational time, which becomes more impor-
tant when dealing with large datasets. The FLOWSOM
[73] algorithm showed the best clustering accuracy and
was one of the fastest methods when applied to large
datasets, with a linear complexity with respect to the
number of cells. CytoCompare [85] is a tool which was
created to perform the comparison of the clustering
results of three methods: SPADE, ViSNE/Accense [82]
and Citrus [86].
The clustering algorithms described above can also
be applied to image derived features, although, as was
the case for visualisation techniques, the high correla-
tion between features might bias clustering results. The
redundancy of the features can be reduced by first
applying a PCA to this type of data, and performing

58
 Expression data

Trajectory
Clustering
inference

Similarities within Similarities between cells

clusters are preserved are preserved and displayed
in lower dimensions

Pseudotime

Fig. 3. In order to identify structures in an expression data matrix, two types of methods can be used. Clustering-based methods will tend
to maximise the similarities between cells within clusters while maximising the differences between clusters. These methods thus help to
identify homogeneous groups of cells in the data. On the other hand, trajectory inference methods will tend to preserve the local similarities
between cells, ordering them along trajectories which represent gradual changes between similar cells.

clustering on the principal components of the PCA. In
scRNA-Seq data, clustering is more tricky because the
gene expression contains noise and the data is very
sparse. Cells may mistakenly be grouped together
based on technical noise attributed to sequencing
depth or library size, rather than actual biological
effects. This raises the need for new tools, which are
able to overcome this issue. Several tools do not com-
pare the expression patterns of cells directly anymore,
but apply tricks to perform more accurate clustering:
SC3 [87] computes a consensus clustering over several
kmeans runs at the cost of a high computational cost,
BackSPIN [88] uses a biclustering method and
DIMM-SC [89] was designed specifically for droplet-
based single-cell RNA seq data.
Another characteristic of scRNA-Seq data is the
high amount of dropout events. Some clustering
methods were specifically designed to deal with this
artefact, either by imputing the expected value of
dropout candidates (CIDR [90]), or by computing the
similarities between cells with techniques that are
robust to dropouts (SIMLR [91], SNN-Cliq [92], SCE-
NIC [93]). The PAGODA [94] algorithm also accounts
for technical biases such as the expression magnitude
and the cell cycle.
Approaches for modelling gradual transitions
Another set of approaches, called trajectory inference
(TI) methods, aim to reconstruct the developmental pro-
cess that cells are undergoing. The resulting trajectory
consists of states and transitions, with each cell mapped
to a pseudotemporal location in the trajectory (Fig. 4A).
Various visualisation techniques can aid in interpreting

59
 A B

State 2

Expression of Marker 3
State 5 State 3

State 4

State 1

C
b) Marker 1 Marker 2 D

Marker 3 Marker 4

Pseudotime

State 1 State 3 State 4

Fig. 4. There are several approaches to visualising trajectory models inferred by TI methods. (A) The most common visualisation is a
dimensionality reduction where similar cells are placed close together. The cells are typically coloured based on prior knowledge (e.g. cell
type) or computationally inferred clustering, and are overlaid by the trajectory inferred by the TI method. (B) A scatter plot can be used to
demonstrate a response in gene expression over pseudotime. (C) Colouring of the cells in the dimensionality reduction plot can also be
used to compare the gene expression profiles. (D) In order to obtain an overview of the dynamics of a large number of genes, these genes
can be grouped together into modules, and one path along the trajectory can be visualised in the form of a heatmap.

the cell state- and branching point delineation, by visual-
ising the expression value of a marker over time
(Fig. 4B), comparing the gene expression values in cells
within the reduced dimensions (Fig. 4C), or grouping
genes together in pseudotemporally coregulated modules
(Fig. 4D). Cannoodt et al. [95] provide an overview of
several commonly used TI methods, organising them by
the different components they are based on.
Trajectory inference was first explored on mass
cytometry in order to reconstruct the differentiation of
hematopoietic stem cells into naive B cells [96]. Since
then, TI methods have been used increasingly to
reconstruct cell developmental trajectories. There are
several strategies TI methods use to tackle this com-
plexity, and the choice of which method is most
appropriate will thereby depend on the characteristics
of the given dataset [97]. Pioneering TI methods were
often specialised in producing a fixed trajectory type
(e.g. linear [96,98], bifurcating [70,99] or cyclical [100]).
Some methods require specific input [101], while others

60
are capable of inferring the trajectory structure in an
unbiased way [72,102]. A recent comparative review
[97] assessed the performance of more than thirty TI
methods on both synthetic and real scRNA-Seq data-
sets, providing useful practical guidelines to choose the
most appropriate methods. Notably, no method con-
sistently outperformed the others on all datasets.
Rather, various sets of methods were better suited to
specific trajectories in the datasets, with some methods
better identifying linear trajectories, and others effi-
ciently identifying cycles. A good practice would there-
fore be to identify a set of TI methods to apply to the
data based on the expected structure, and comparing
the results of at least 2–3 methods to confirm the bio-
logical findings.
Differential analysis
Cytometry-based approaches
In order to identify cell populations which differ
between different experimental conditions (e.g between
samples of patients with different clinical outcomes),
cytometry data can first be clustered, and these clusters
can be compared between the conditions. In FLOWSOM
[73], the user can provide a fold-change threshold, to
colour clusters which differ between the conditions.
The Citrus [86] and COMPASS [103] algorithms both
perform model selection to identify the clusters which
are best associated with a certain condition. A similar
method was implemented, which groups cells into
hyperspheres instead of clusters (Cydar [59]). Convolu-
tional neural networks have also been used to identify
subpopulations of cells which differ the most between
two conditions (CellCNN [104]). However, none of
these methods directly cope with complex experiments
and may therefore be sensitive to batch effects, which
might be misinterpreted as the main difference between
the conditions. One solution is to first remove possible
batch effects in a preprocessing step before performing
differential analysis. A CYTOF workflow [105] has
been proposed, which first applies clustering and then
uses Gaussian linear mixture models to perform differ-
ential analysis while accounting for possible batch
effect, paired experiments and other sources of techni-
cal variance in the data.
Sequencing-based approaches
The technical biases which have to be dealt with are
even larger in single-cell and bulk RNA-Seq data, as
many genes are lowly expressed and noisy. Several
methods were proposed to specifically tackle differential
expression (DE) of genes in scRNA-Seq data (SCDE
[106], MAST [107], scDD [108]). These methods use
mixture models or Bayesian modelling frameworks to
identify both the technical effects between samples
(mainly caused by the gene detection rate) and the vari-
ance which is related to the condition being tested.
Another method, CENSUS [72], normalises the single-
cell gene expression into relative transcript counts
(accounting for technical variability between cells) in
time series studies specifically, allowing for the identifi-
cation of genes whose expression varies along time.
These single-cell specific DE methods aim to free them-
selves from the idea that gene expression is unimodal
across cells. Indeed, as many cells often show unmea-
sured genes, either due to biological or technical effects,
these methods model gene expression through more
elaborate distributions.
However, a recent study [109], which compared 36
differential gene expression approaches, concluded that
methods that were largely used for the DE analysis of
bulk RNA datasets (such as DESEQ2 [110], edger [111],
VOOM [112]), were in fact not performing worse than
single-cell specific DE methods on scRNA-Seq data-
sets. Single-cell specific DE approaches also required
more computational time, although they scaled well
with increasing cell numbers. This comparative study
highlighted the fact that an important trend that gen-
erally improved a DE analysis results was accurate
gene filtering, which reduces noise in lowly expressed
genes, leading to less false positive genes being identi-
fied as differentially expressed.
Advanced computational approaches
Network inference
Single-cell transcriptomics provide a rich source of
data, by quantifying the expression profiles of thou-
sands of cells. The intercellular heterogeneity which
naturally results from biological stochasticity [113]
allows inferring mechanisms of gene regulation involv-
ing transcription factors and their target genes. More
complex, nonlinear interactions between genes can be
studied at the single-cell level, as was shown with the
PIDC [114] algorithm, which was able to infer regula-
tory networks involved in developmental processes
from sc-qPCR datasets. However, inferring one global
regulatory network from thousands of cells might not
always prove accurate. Different subpopulations of
cells in the data might be undergoing different regula-
tory processes, which is why some methods were
implemented specifically to compute differential regu-
latory networks. These methods derive one regulatory
61
 network for each cell subtype (CSRF [115], P olya tree
models [116]).
In order to improve the inference of gene regulatory
networks, external sources of information can be pro-
vided. As was discussed in the section ‘Approaches
modelling gradual transitions’, cells can be ordered
along developmental trajectories. Some network infer-
ence methods can include the information from these
inferred trajectories to reconstruct dynamic regulatory
networks (AR1MA1 [117], SCODE [118]). Another
source of external information could come from pertur-
bational studies, in which genes are knocked out and
the consequences on the transcriptome can be observed
[21]. New tools will be needed to optimally use this type
of data in order to infer regulatory networks.
Single-cell transcriptomics data represent a rich
source of information to infer interactions which occur
between genes and transcription factors. However, new
studies are highlighting the need to not only focus on
a single-cell’s transcripts, but also the methylation
state of the DNA, the chromatin state and other epige-
nomic data that might enrich our knowledge of the
gene regulation dynamics [119,120].
Single-cell multi-omics data integration
Single-cell transcriptomics, proteomics, genomics and
epigenomics have provided a level of understanding of
the cellular heterogeneity that could not be reached
with bulk studies. However, the models which are
inferred from single technologies are by definition
incomplete. Indeed, the relationships between the gen-
ome, the amount of transcripts and proteins in a single
cell are not always straightforward. Transcriptional
regulatory mechanisms such as methylation may for
instance alter the correlation between the gene copy
number and the associated number of transcripts.
Moreover, post-transcriptional mechanisms regulating
protein translation and stability may also influence the
relation between the number of transcripts and pro-
teins in a cell. In order to fully understand and to start
modelling the mechanisms involved in single cells, it
will therefore be essential to integrate complementary
types of data from the same single cells [26].
New experimental approaches have already been able
to achieve a simultaneous and multiparameter measure-
ment by combining methods. The study of the genome
together with the transcriptome [24,121] for instance has
confirmed the existence of a strong correlation between
genes with high copy numbers and the number of
mRNA transcripts. The joint analysis of the methylome
together with the transcriptome [25] also corroborated
the negative relation between the methylation of a gene
62
and its transcription. More surprisingly, the measure-
ment of both transcripts and proteins [122,123] in single
cells has highlighted the fact that the amount of these
two entities was poorly correlated. This could be due to
the fact that transcription occurs in bursts, resulting in
high discrepancies between the numbers of transcripts,
whereas protein levels have been shown to be more
stable for particular genes [124].
The experimental procedures cited above led to low-
throughput datasets, typically containing 100 cells at
most, and could therefore be analysed by regular corre-
lation studies to assess the links between different omics
entities. The recently published CITE-seq [22] and
REAP-seq [23] methods have allowed the simultaneous
measurement of the transcriptome as well as 100 pro-
teins in thousands of cells, and have the potential to
measure thousands of proteins in single cells, as these
proteins are tagged with synthetic oligonucleotides.
Some studies have also achieved a broader characterisa-
tion of single cells by combining proteomics- and imag-
ing-based approaches [125,126]. As new experimental
procedures keep providing larger and larger datasets,
and new tools allow getting more insight into the mech-
anisms of regulations at the single-cell level [127,128],
there is a great need for multi-omics integrative compu-
tational tools. These tools should have the ability to
combine the information coming from complementary
sources to infer complex global models.
Conclusions and future perspectives
Various high-throughput approaches currently allow
studying cell populations into unprecedented depth.
The rapid development of novel technologies or
hybridisations between them is generating large and
complex datasets that require designing novel computa-
tional approaches for preprocessing, visualising and
extracting novel patterns from them. As novel tech-
nologies arise, the development of computational tools
and the adequate benchmarking between them is lag-
ging behind. Indeed, many computational approaches
to study single-cell data are continuously being pub-
lished, but the number of benchmark studies that objec-
tively compare these methods is under-represented.
Nevertheless, such benchmarks are essential to extract
useful guidelines for biologists who want to use these
tools, pinpoint limitations of current approaches and
highlight novel directions for future tool development.
While current methods mainly focus on cells in sus-
pension, novel advances that include the spatial con-
text will stimulate novel classes of computational tools
that will enable modelling cellular interactions and cell
dynamics into much greater depth. Such techniques
will allow going from cells in isolation to tissues and
organs, offering new perspectives for multiscale mod-
elling. On the other hand, single-cell multi-omics
approaches are providing complementary information
that can relate epigenetic, transcriptional and transla-
tional information, paving the way for single-cell mul-
ti-omics and multi-source data integration.
All of these advances strengthen the idea that the
life sciences are becoming even more data-driven
sciences. To be able to analyse and correctly interpret
the results of computational pipelines, young research-
ers thus should be trained adequately in properly using
and understanding the principles of these novel com-
putational approaches.
Acknowledgements
We thank Sofie Van Gassen, Robrecht Cannoodt,
Niels Vandamme and Daniel Peralta for critical com-
ments and valuable input. HT is funded by a BOF-
IOP grant from Ghent University; YS is an ISAC
Marylou Ingram scholar.
Conflict of interest
The authors declare no competing interests.
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