representation bisulfite sequencing (scRRBS-seq) [52]. The second
one, post-bisulfite adaptor tagging (PBAT) [53], was implemented
on single cells [51]. The third one implemented another variant of
the post-bisulfite library construction procedure, deriving lower
coverage while using lower sequencing depth [54]. Importantly,
alternative approaches for characterizing DNA methylation are also
applicable at the single-cell level. In the single-cell restriction anal-
ysis of methylation (SCRAM) approach [55], methylation-sensitive
restriction enzymes and single-cell quantitative PCR (qPCR) are
combined to facilitate profiling of methylation state across small
panels of target sites.1.4.2 Single-Cell Histone
Modifications
Different from DNA methylation that can be measured directly
from bisulfite-converted sequences, histone modifications are pro-
filed indirectly using chromatin immunoprecipitation followed by
sequencing (ChIP-seq). The measures of histone modifications are
defined by the enrichment of reads by distinguishing statically true
positive from the genomic background when sequencing binding
peaks at adequate depth. It is more problematic if the same
approaches are applied to single cells, since the absence of the
statistical pooling disables the direct way to distinguish true posi-
tives from false positives. A similar effect may restrict the single-cell
application of techniques for mapping accessibility patterns using
DNase [56], micrococcal nuclease (MNase) [57], or transposases
(ATAC-seq) [58]. Nevertheless, some technologies are under
development to tolerate high number of false-positive readouts by
multiplexing analysis of thousands of single cells [59–62]. Alterna-
tively, pooling data of related loci (e.g., clustering of loci) can be
used to derive higher-quality data on generalized single-cell epige-
nomic features, even if the quality of individual profiles is not
optimal [63, 64]. Therefore, in summary, genome-wide single-
cell histone modification or TF-binding mapping is still under
development, which is far behind the single-cell RNA-seq.1.4.3 Single-Cell
Chromosomal Confirmation
Capture
Chromosome conformation capture (3C) and derivative techni-
ques [65–67], such as Hi-C, allow characterization of chromo-
somal topologies at different size scales: from whole
chromosomes to topologically associating domains (TADs) and
down to sub-megabase-scale chromosomal loops. Since it is clear
as early as from microscopy that chromosome folding is highly
nuclei-specific [68, 69], understanding large-scale chromosomal
structure inevitably involves considerations of single-cell dynamics.
The ability to scale up 3C toward the sequencing of billions of
ligation products makes Hi-C on cell populations a powerful exper-
imental tool. Similarly, Hi-C can be scaled down effectively to
single nuclei, as it encodes distinct ligation events within individual
nuclei if an appropriate nuclei separation and labeling scheme isApplications of Single-Cell Sequencing for Multiomics 333