1.3 Sequencing
Genomes of Single
Cells
Although bulk approaches for studying genetic variation have iden-
tified thousands of new unicellular species and determined genetic
etiologies for thousands of human disease, most of them have been
done at the level of the ecosystem or organism. We know that the
cell-to-cell heterogeneity exists not only in transcriptome but also
in genome, i.e., the genomes within the cells of an individual
multicellular organism are not always the same. Single-cell geno-
mics aims to provide new perspectives to our understanding of
genetics by bringing the study of genomes to the cellular level.
Sequencing a genome of single cells has four steps, which remain
technically challenging: (1) efficient physical isolation of individual
cells, (2) amplification of the genome of that single cell to acquire
sufficient material for downstream analyses, (3) querying the
genome in a cost-effective manner to identify variation that can
test the hypotheses of the study, and (4) interpreting the data
within the context of biases and error that are introduced during
the first three steps. Reference2 gives a comprehensive review of
the state of this field.1.4 Sequencing
Epigenomes of Single
Cells
Epigenome is an integrative collection of chemical modifications,
associations, and conformations of genomic DNA sequences,
including histone modifications and variations, DNA methylations,
nucleosome positioning, chromatin conformations, etc. The epige-
nomics aims to link these with epigenetic memory, cellular identity,
and tissue-specific functions. While the average epigenomic features
across large cell populations have been largely characterized with
the help of current techniques, the tissue complexity and cell-to-cell
heterogeneity are driving the development of single-cell epige-
nomics. We, here, survey emerging methodologies for single-cell
epigenomics, which have been comprehensively depicted in Fig. 1
of ref.49.1.4.1 Single-Cell DNA
Methylation
DNA methylation is an epigenetic mechanism that occurs by the
addition of a methyl (CH3) group to DNA, thereby often modify-
ing the function of the genes. The most widely characterized DNA
methylation process is the covalent addition of the methyl group at
the 5-carbon of the cytosine of the dinucleotide CpG, resulting in
5-methylcytosine (5-mC). For DNA methylation profiling of bulk
tissue or cell populations, bisulfite sequencing has enabled high-
throughput interrogation of CpG modifications, by generating
millions or billions of reads to accurately define the methylation
state across the entire genome or within some strategic genomic
regions [50]. Single-cell bisulfite sequencing improves our under-
standing of the methylome intrapopulation distribution by simul-
taneously defining the methylation states of CpGs within an entire
epigenome. Three single-cell methylome assays based on bisulfite
sequencing have been reported recently. The first ones include
single-cell bisulfite sequencing (scBS-seq) [51] and reduced332 Yungang Xu and Xiaobo Zhou