plate/chip, a process known as multiplexing. To avoid the chaos that would result
from simply mixing heterogeneous sources of DNA together and trying to assign
each read back to its correct source, prior to mixing, each DNA library can be ligated
to adapter primers that are identical except for a short region (usually 2–5 nt) deemed
an index or bar code. Ligating a different indexed adaptor(s) to each sample allows
the resulting sequence reads from the mixture to be correctly deconvoluted and
assigned to their appropriate source (via simple in silico sorting). If both the forward
and reverse adapters contain indices (called “double indexing”), deconvolution of
data is significantly more accurate (Kircher et al. 2012).
3.23 Phasing
Phasing is the term used to describe assignment of alleles in a diploid individual(s)
into haplotypes (Roach et al. 2011). With regard to genome sequencing, certain
types of phasing can be used to circumvent assembly problems associated with
heterozygosity. As discussed above, starting with a genetically homogeneous individual or a haploid tissue is an ideal way to start a genome sequencing project.
However, homogeneity is not always an option, especially when dealing with
obligate outcrossing species. The “gold standard” phasing technique used in genome
sequencing is called trio phasing. For an individual produced by sexual reproduction, the genomes of that individual’s parents are sequenced via a second-generation
(i.e., short-read) method (e.g., Illumina sequencing). The offspring’s DNA is
sequenced using a third-generation (long-read) instrument. K-mers from both parents are compared, and those unique to the maternal parent are matched with the
reads of the child. Those reads recognized by the maternal k-mers are considered part
of the maternal haplotype. The same process is repeated using unique k-mers from
the father. Further assembly of the maternal and paternal haplotypes into contigs/
scaffolds is then conducted to produce two haplotype assemblies.
3.24 Hi-C
The fact that interphase chromosomes occupy distinct domains within nuclei has
been exploited as a means of looking at chromatin organization and determining
which DNA sequences are typically near each other in the 3D space of the nucleus. A
chromatin conformation capture technique known as Hi-C has become a popular
high-throughput means of exploring chromatin relationships (Denker and De Laat
2016). The Hi-C concept/method is illustrated in Fig. 9. In brief, nuclei are treated
with formaldehyde which introduces cross-linking between adjacent chromatin
regions. Ligation of the DNA molecules from the cross-linked regions produces
chimeric DNA molecules that can be sequenced with second- or third-generation
sequencing. If two sequences are found associated with each other more than one
Sequencing Plant Genomes
143
from simply mixing heterogeneous sources of DNA together and trying to assign
each read back to its correct source, prior to mixing, each DNA library can be ligated
to adapter primers that are identical except for a short region (usually 2–5 nt) deemed
an index or bar code. Ligating a different indexed adaptor(s) to each sample allows
the resulting sequence reads from the mixture to be correctly deconvoluted and
assigned to their appropriate source (via simple in silico sorting). If both the forward
and reverse adapters contain indices (called “double indexing”), deconvolution of
data is significantly more accurate (Kircher et al. 2012).
3.23 Phasing
Phasing is the term used to describe assignment of alleles in a diploid individual(s)
into haplotypes (Roach et al. 2011). With regard to genome sequencing, certain
types of phasing can be used to circumvent assembly problems associated with
heterozygosity. As discussed above, starting with a genetically homogeneous individual or a haploid tissue is an ideal way to start a genome sequencing project.
However, homogeneity is not always an option, especially when dealing with
obligate outcrossing species. The “gold standard” phasing technique used in genome
sequencing is called trio phasing. For an individual produced by sexual reproduction, the genomes of that individual’s parents are sequenced via a second-generation
(i.e., short-read) method (e.g., Illumina sequencing). The offspring’s DNA is
sequenced using a third-generation (long-read) instrument. K-mers from both parents are compared, and those unique to the maternal parent are matched with the
reads of the child. Those reads recognized by the maternal k-mers are considered part
of the maternal haplotype. The same process is repeated using unique k-mers from
the father. Further assembly of the maternal and paternal haplotypes into contigs/
scaffolds is then conducted to produce two haplotype assemblies.
3.24 Hi-C
The fact that interphase chromosomes occupy distinct domains within nuclei has
been exploited as a means of looking at chromatin organization and determining
which DNA sequences are typically near each other in the 3D space of the nucleus. A
chromatin conformation capture technique known as Hi-C has become a popular
high-throughput means of exploring chromatin relationships (Denker and De Laat
2016). The Hi-C concept/method is illustrated in Fig. 9. In brief, nuclei are treated
with formaldehyde which introduces cross-linking between adjacent chromatin
regions. Ligation of the DNA molecules from the cross-linked regions produces
chimeric DNA molecules that can be sequenced with second- or third-generation
sequencing. If two sequences are found associated with each other more than one
Sequencing Plant Genomes
143