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2. INTERNATIONAL PARIS
CONGRESS ON AGRICULTURE
& ANIMAL HUSBANDRY
October 24-26, 2023
ISBN: 978-1-955094-54-2
EDITOR
Assoc. Prof. Dr. Nizamettin TURAN
2. INTERNATIONAL PARIS CONGRESS ON
AGRICULTURE & ANIMAL HUSBANDRY
October 24-26, 2023 -Paris
EDITOR
Assoc. Prof. Dr. Nizamettin TURAN
Copyright © Liberty
12.11.2023
by Liberty Academic Publishers
New York, USA
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© Liberty Academic Publishers 2023
The digital PDF version of this title is available Open Access and distributed under the terms
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derivative works do not need to be licensed on the same terms.
ISBN: 978-1-955094-54-2
77
PLANT EPIGENETIC RESEARCH: CONTRASTING WGBS WITH BISEQ
Prof. Dr. Ayse Gul INCE
Vocational School of Technical Sciences, Akdeniz University, 07070 Antalya, Turkey
ORCID NO: https://orcid.org/0000-0002-9015-6580
Prof. Dr. Mehmet KARACA
Department of Field Crops, Faculty of Agriculture, Akdeniz University, Antalya, 07059
Turkey
ORCID NO: https://orcid.org/0000-0003-3219-9109
ABSTRACT
The genome employs both epigenetic and genetic pathways to generate responses to various
internal and external stimuli. Epigenesis refers to the process by which chemical modifications
occur in the genome, as well as changes in chromatin structure, that are independent of
alterations in the DNA sequence. DNA cytosine methylation is a crucial epigenetic mechanism
that regulates all genetic processes, including gene transcription and transposition, DNA
replication and repair, cell differentiation, gene silencing, and imprinting. It also influences
vernalization, heterosis, drought and salt tolerance, biodefense, transgenic expression, and the
expression of foreign DNA in cells. Cytosine methylation is a prevalent occurrence in plants,
typically shown in three distinct sequences. These sequences are CG, CHG, and CHH (where
H represents A, C, or T). Two approaches are often used to study DNA cytosine methylation.
WGBS is a second next-generation sequencing technique, and BiSeq was initiated using the
Sanger sequencing. We employed BiSeq in a number of plant species, including cotton, and
we are preparing to apply the WGBS approach to study plant epigenetics. In this work, we
compared benefits and drawbacks of the WGBS and BiSeq approaches. In summary, both of
the methods start with the use of sodium bisulfite treatment of genomic DNA, the number of
targets greatly differ, BiSeq usually involves in cloning and Sanger sequencing. The process
of WGBS begins with the random fragmentation of DNA into tiny pieces in the presence of a
spike, to which adapters are ligated. NGS technologies are used to sequence size-selected
library fragments. Despite having significant differences, these two sequencing methods might
be applied to the same study. With the aid of tables and figures, we address a few technical
features of these bisulfite sequencing techniques in the current work.
Keywords: BiSeq, 5-methylcytosine, methylation, NGS, PCR, sequencing
78
INTRODUCTION
Today, technological advancements have presented unparalleled prospects for the surveillance
of chromatin changes, gene expression, and genomic structure. The initial documentation of
various conventional epigenetic phenomena, like as transposable element inactivation,
imprinting, paramutation, transgene silencing, and co-suppression, primarily originated from
studies conducted on plants. The integration of classical genetic investigations with recently
developed sequencing technology has enabled the examination of several epigenetic
phenomena with an unprecedented level of precision, a capability that was previously
inconceivable until recent years. The current period presents a stimulating opportunity to
engage in the research of plant epigenetics (Olkhov-Mitsel & Bapat, 2012; Hernandez et al.,
2013; Kurdyukov & Bullock, 2016; Beck et al., 2021; Ince & Karaca, 2021).
The term 'epigenetic' pertains to heritable patterns of gene expression that are determined
by the gene's DNA sequence. The investigation of plants has yielded a multitude of significant
contributions to the discipline of epigenetics. The prevalence of epigenetic regulation in plants
can be attributed to their specific patterns of development, lifestyle characteristics, and
evolutionary trajectory. Plants rely significantly on alterations in gene expression to effectively
react to environmental stimuli, and it is highly probable that the regulation of gene expression
by chromatin changes plays a pivotal role in these responses. Furthermore, it has been shown
that plants have a decreased level of chromatin "resetting" during the process of sexual
reproduction in comparison to animals. This particular trait has the potential to facilitate the
transfer of epimutations that are acquired over the lifespan of plants. Moreover, a significant
proportion of plant species has the capacity for asexual reproduction, leading to the generation
of vegetative clones. The mechanism presents promising pathways for the propagation of
epigenetic states via mitotic inheritance, finally culminating in the emergence of notable
characteristics. The investigation of epigenetics in plants holds significant scholarly
significance (Kurdyukov & Bullock, 2016; Ince & Karaca, 2021; Karaca & Ince 2023; Van
Antro et al., 2023).
The genome employs both epigenetic and genetic pathways to generate responses to
various internal and external stimuli. Epigenesis refers to the process by which chemical
modifications occur in the genome, as well as changes in chromatin structure, that are
independent of alterations in the DNA sequence. These modifications and structural changes
are a consequence of cells or tissues detecting and responding to signals generated by
environmental factors or internal signals. The resulting information is then encoded in the
epigenome. The primary hypothesis posited in the project proposal asserts that the genome
effect plays a crucial role in influencing the epigenome. Furthermore, it is postulated that the
manipulation of the epigenome is feasible, and that valuable insights into the fundamental
principles of epigenome engineering can be accumulated. The ramifications of epigenesis
provide challenges of an intricate kind due to its deviation from the criteria outlined in the
modified central dogma of molecular biology (Ince & Karaca, 2021; Sun et al., 2022; Agius et
al., 2023; Karaca & Ince 2023; Van Antro et al., 2023).
The responsibilities of a genome encompass generating a comprehensive reaction to
various internal and external stimuli, overseeing the functioning of the host cell through this
response, and, when required, safeguarding and transmitting genetic material to ensure the
79
perpetuation of future generations. The functions carried out by the genome are accomplished
through the utilization of both epigenetic and genetic mechanisms (Ahn et al., 2017; Crespo-
Salvador et al., 2018). The process of epigenesis is believed to encompass a sequence of cellular
processes that can give rise to a novel phenotype. This occurs through chemical modifications
that occur independently of alterations in the DNA sequence, as well as changes in the structure
of chromatin. These modifications are triggered by signals originating from either the
environment or internal factors, which are detected by the cell or tissue and subsequently elicit
a response (Gapper et al., 2014).
The epigenetic result is determined by the presence of epialleles. An epiallele refers to a
heritable variation in chromatin structure that results in a persistent allelic difference.
According to Eichten et al. (2014), classification of epialleles can be categorized into three
distinct states: pure, selective (facilitate), and obligate (obligate) epialleles. The pure epiallelic
state is characterized as an epiallelic condition that is wholly autonomous from genetic
information, whereas the obligatory epiallelic state is contingent upon genetic variation. The
condition of facultative epiallele status refers to the situation in which genetic variation has a
role in determining the alternative chromatin state. The term "chromatin state" encompasses
various components such as histone variations, histone modifications (such as methylation and
acetylation), DNA methylation, and impacts and modifications of small RNA molecules
(Eichten et al., 2014).
DNA methylation is a prominent method of epigenetic diversity in eukaryotic genomes. It
involves the enzymatic alteration of DNA and chromatin. Special methyltransferases could
methylate nuclear DNA (nDNA). The predominant form of DNA methylation involves the
addition of a methyl group (-CH3) to the C5 site of cytosine. Cytosine methylation is a
prevalent occurrence in plants, typically shown in three distinct sequences. These sequences
are CG, CHG, and CHH (where H represents A, C, or T). The process of DNA
hypermethylation, mediated by methylase enzymes, has been found to be associated with the
suppression of gene expression in promoter regions, but it is associated with gene activation in
coding sequences (Ince & Karaca, 2021; Karaca & Ince, 2023).
DNA cytosine methylation has a role in the activation of transcription and the regulation
of gene expression after transcription. The condition of methylation is characterized by its
dynamic nature, as the actions of methyltransferases can be counteracted by demethylases,
hence allowing for reversibility. In addition to protein enzymes, short RNAs have a role in the
process of DNA methylation through the mechanism known as RNA-guided DNA methylation
(RdDM) (Yaari et al., 2019).
This article provides a concise introduction to DNA methylation determination methods
and compares the merits and drawbacks of bisulfite sequencing with whole genome sequencing
techniques.
80
METHODS FOR DNA METHYLATION ANALYSIS
Currently, a range of techniques are employed to address global and gene-specific cytosine
methylation (Figure 1). Methods for analysing DNA methylation in research typically involve
the use of restriction enzyme-based, bisulfite-based, affinity-based, or a combination of these
methodologies. The determination of methylation can be categorized into three main
methodologies, which include the following methods: (i) Bisulfite-based: Whole genome
bisulfite sequencing, Reduced representation bisulfite sequencing, Massively Clonal
sequencing platforms, Denaturing HPLC, Bisulfite methylation profiling, Bisulfite sequencing,
Bisulfite padlock probes (BSPP), Pyrosequencing, Methylation-specific PCR (MSP),
Methylation sensitive melting curve analysis (MS-MCA), Methylation sensitive high
resolution melting (MS-HRM), Sensitive melting analysis after real-time (SMART)-MSP,
Methylation-specific fluorescent amplicon generation (MS-FLAG), Methylation sensitive
single nucleotide primer extension (MS-SNuPE), (ii) Affinity-based: Methylated DNA
immunoprecipitation (MeDIP), Methylated CpG island recovery assay (MIRA), Methyl
binding domain proteins MBD column chromatography, MeDIP-PCR, (iii) Restriction
enzymes-based: Restriction landmark genome scanning (RLGS), HpaII tiny fragment
Enrichment by Ligation-mediated PCR (HELP), Luminometric methylation assay, Methyl-
Seq, Methylation sensitive cut counting (MSCC), Methylated CpG island amplification
(MCA), Methylation amplification DNA chip (MAD) and promoter-associated MAD
(PMAD), Comprehensive high-throughput arrays for relative methylation (CHARM),
Microarray-based methylation assessment of single samples (MMASS), Methylscope,
Methylation hybridization (DMH), Methylation SNP (MSNP), Methylation sensitive arbitrary-
primed PCR (MS-AP-PCR), Amplification of intermethylated sites (AIMS), TD-MS-RAPD-
PCR. Some of these are shown in Figure 1 and Figure 2 according to their usage strategies.
Typing and profiling of DNA methylation with these technologies are suitable for high-
throughput applications that encompass established and new methods used for (A) identifying
DNA methylation and (B) confirming DNA methylation-based biomarkers, while outlining
their primary benefits and constraints (Laird, 2010; Olkhov-Mitsel & Bapat, 2012; Karaca et
al., 2019; Agius et al., 2023).
The primary consideration is that the selected methodology must provide an objective
response to the biological inquiry posed by the researcher. Nevertheless, it is imperative to
consider various additional crucial factors in the selection of a DNA methylation analysis
method. These factors encompass the objectives of the study, such as the identification of
newly occurring epigenetic alterations or the examination of established methylation sites in
particular genes of interest. Additionally, the quantity and quality of the DNA sample(s), the
desired level of sensitivity and specificity, the reliability and ease of use of the method, the
accessibility of bioinformatics software for data analysis and interpretation, the availability of
specialized equipment and reagents, as well as the associated costs, all play a significant role
in the decision-making process. The researcher can select from different ways based on
whether they are studying a gene that is already known or one that is unknown. According to
Kurdyukov & Bullock (2016), these methods can be organized and presented in Figure 2.
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Figure 1. Some methods for DNA methylation analysis.
Figure 2. Algorithm for selecting an appropriate approach for analysing DNA methylation
adapted from Kurdyukov & Bullock (2016).
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Determination of Differentially Methylated Regions and Differentially Methylated
Cytosine
The techniques outlined in this article can be employed to ascertain the global alterations
in the DNA methylation state of the sample(s) under investigation. Nevertheless, “what is the
process of identifying and evaluating particular genes/regulatory areas of interest that exhibit
differential methylation?” is an important question. Bisulfite sequencing and whole genome
bisulfite sequencing are crucial techniques used to identify Differentially Methylated Regions
or Differentially Methylated Cytosine (Karaca & Ince, 2023).
Bisulfite Sequencing (BiSeq)
Existing DNA sequencing technologies lack the capability to differentiate between methyl
cytosine and cytosine. The bisulfite-assisted deamination method is widely recognized as the
gold standard technique for detecting DNA cytosine methylation (Baumann and Doerge, 2011).
This method allows for the determination of the methylation status of individual cytosine
nucleotides over the entire genome, making it particularly well-suited for large-scale DNA
sequencing approaches. Following the sulfonation events carried out by methyl particles,
cytosine sulfonate is formed through bisulfite interaction at the carbon of the cytosine base.
Subsequently, the emergence of ammonium takes place, accompanied by deamination breaks.
The formation of uracil sulphonate occurs as a consequence of this reaction. During the final
process, desulfination reactions occur, resulting in the formation of uracil through the removal
of bisulfite (HSO3) from uracil sulfate. The conversion of uracil within the array is achieved
through the utilization of polymerase chain reaction (PCR) (Figure 3). These transformed
residues are then interpreted as thymine using PCR-amplification and subsequent Sanger
sequencing analysis (Kurdyukov & Bullock 2016; Ince & Karaca, 2021; Karaca & Ince, 2023).
Figure 3. Bisulfite sequencing overview.
In this method, bisulfite application is made after genomic DNA isolation. PCR is
performed using bisulfite specific primers. After the PCR process, agarose gel purification is
performed. Purified amplicons are transformed and cloned. Finally, DNA sequence is
determined (Figure 3). There are several programs such as, KisMeth, CyMATE, etc., for
methylation analysis in bisulfite sequencing methods (Ince & Karaca, 2021).
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Whole Genome Bisulfite Sequencing (WGBS)
Whole-genome bisulfite sequencing (WGBS), also known as MethylC-seq, is a technique that
has the potential to examine every cytosine in the genome at the individual nucleotide level.
The WGBS approach allows for the analysis of methylation in all CG, CHG, and CHH context
sequences in a genome by examining all the cytosines. WGBS is analogous to whole genome
sequencing, except for the bisulfite conversion step. This method is the most thorough among
all the ways now in existence. The sole constraints lie in the expenses and challenges associated
with the analysis of NGS data. As previously stated, cytosine nucleotides that are not
methylated are converted to thymine nucleotides following bisulfite treatment, and assembling
DNA consisting of only three bases is extremely challenging.
This procedure entails the extraction of genomic DNA, followed by enzymatic or physical
fragmentation (such as sonication or passage through microscopic pores) into segments
measuring 200-300 nucleotides in length. Subsequently, the process of bisulfite conversion
and cleaning can be carried out. After treating with bisulfite, the fragments are cleaved at the
ends, and a single adenine (deoxyadenine) nucleotide is appended and linked to the fragment
ends using specific adapters and index sequences. The selection technique relies on the lengths
of the fragments, exclusively choosing fragments that possess distinct adapters at both ends.
Subsequently, bridge PCR can be performed. The libraries produced are then analyzed using
an appropriate next-generation DNA sequencing technique (Figure 4) (Gong et al., 2022; Agius
et al., 2023). Recently, this method has been employed in applications such as Adaptase,
TruSeq, and SLAT methods.
Figure 4. Whole Genome Bisulfite Sequencing steps.
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Various programs, including B-SOLONA, BatMeth, BiQ Analyzer HT, BiSeq, BISMA,
Bismark, Bis-SNP, Bisulfighter, BRAT, BSMAP, BS-Seeker, DMAP, GSNAP, GBSA, Last,
MOABS, MethylExtract, MethylSig, RMAP, SAAP-RRBS, etc., are available for methylation
analysis in whole genome bisulfite sequencing (Olkhov-Mitsel & Bapat, 2012; Kurdyukov &
Bullock, 2016; Gong et al., 2022; Agius et al., 2023).
CONTRASTING WGBS WITH BISEQ
Bisulfite sequencing method compared to whole genome bisulfite sequencing method, bisulfite
sequencing offers the benefit of furnishing more comprehensive and intricate data pertaining
to gene body and regulator sequences. Conversely, NGS-based techniques often prioritize
depth, coverage, and accuracy. Whole genome bisulfite sequencing method exhibits superior
coverage compared to other techniques. In the WGBS method, it is optimal to have a depth of
30 times or greater (Olkhov-Mitsel & Bapat, 2012; Kurdyukov & Bullock, 2016; Gong et al.,
2022; Agius et al., 2023).
When the bisulfite sequencing method is examined, the utilization of bisulfite sequencing
can present difficulties. Bisulfite PCR leads to challenges in the functioning of target areas,
resulting in difficulty caused by BiSeq. Bisulfite conversion decreases the complexity of the
genome to only three nucleotides, making post sequence alignment more challenging.
Furthermore, the process of bisulfite conversion causes DNA to break apart, which, together
with reduced complexity, hinders the amplification of large fragments and may potentially
produce hybrid products. Ensuring the full conversion of non-methylated cytosines is of utmost
importance, as the predicted degree of DNA methylation relies on it. Hence, it is crucial to
include measures to regulate bisulfite reactions and to closely monitor the presence of cytosines
in non-CpG locations during sequencing, as this serves as an indication of inadequate
conversion. The BiSeq method exhibits greater sensitivity to errors caused by PCR compared
to the WGBS method. For a technological standpoint, the BiSeq approach is appropriate for
research and laboratories of a smaller scale. Due to the use of purification, transformation,
cloning, Sanger DNA sequencing method in the BiSeq method, the study of a low number of
genome regions increases the cost of this method (Olkhov-Mitsel & Bapat, 2012; Kurdyukov
& Bullock, 2016; Gong et al., 2022; Agius et al., 2023).
The WGBS method is well-suited for laboratories that are both large in scale and complex
in nature. The WGBS is a technique that incurs a rather substantial financial investment.
Nevertheless, the utilization of WGBS techniques enables the accurate determination of the
methylation status of both DNA strands at a resolution of individual bases inside a particular
cell or genome, pertaining to a certain stage of development or physiological condition.
Furthermore, the utilization of this approach is favoured owing to the ample availability of
bioinformatic tools that may aid in the analysis and understanding of WGBS data (Crary-
Dooley et al., 2017; Yaari et al., 2019; Shepherd et al., 2022). A previous constraint of WGBS
was the need for a substantial quantity of DNA. However, a modification to the protocol, which
involved delaying the adaptor ligation step until after bisulfite treatment, enabled the routine
use of WGBS with approximately 30 ng of DNA, and in certain instances, as little as 125 pg.
Nevertheless, due to the limited proportion of the genome that can undergo differential
methylation, WGBS is typically unnecessary. Sequencing the part of the genome enriched with
5-methylcytosine (5 mC) is not only a cost-effective method, but it also enables the
85
enhancement of sequencing coverage and, consequently, improves the accuracy in identifying
differentially methylated areas. Sequencing can be performed using any available NGS
technology. Illumina and Life Technologies provide kits specifically designed for this type of
study (Tse et al., 2021; Agius et al., 2023).
Compared to the BiSeq method, WGBS method requires a bioinformatics expert and
appropriate software programs for its work. While both WGBS and BiSeq post-sequencing
analysis share similarities, WGBS analysis necessitates a significant demand for in silico
storage capacity due to the exceptionally large volume of data. The utilization of infrastructure
and bioinformatics expertise, as well as the storage and transfer of data, contribute to the
elevated cost of the WGBS sequencing method. The presence of repeated genomic regions and
an abundance of paralogous and analogous sequences diminishes the precision of analysis in
WGBS. The abundance of samples with diverse origins poses additional technical challenges
in the WGBS method. Moreover, the feasibility of WGBS in small-scale studies is exceedingly
limited (Olkhov-Mitsel & Bapat, 2012; Kurdyukov & Bullock, 2016; Gong et al., 2022; Agius
et al., 2023).
In the single-molecule real-time sequencing (SMRT) method, the sequence of a single
DNA molecule can be extracted using fluorescently labelled nucleotides. Some advantages of
the SMRT method in methylation analysis are (i) no need for chemical modification, (ii) no
need for DNA amplification, (iii) the amount of DNA required is small, and (iv) the DNA
sequence of very long DNA molecules can be extracted.
Nanopore Sequencing is the method in which DNA sequencing is performed by utilizing
the ion current strength specific to each nucleotide while passing single-stranded DNA through
pores formed from proteins with the help of phage DNA polymerase enzyme, without the need
for PCR application. With this method, normal cytosine can be separated from 5-methyl
cytosine, as well as other types of modifications (for example, 5-hydroxymethyl cytosine) can
be determined. However, this method needs a little more time to be established and reduce the
cost.
Two types of algorithms are commonly used in the analysis of bisulfite converted DNA
sequences. These algorithms are free card and three-character algorithms. In free card
alignment, all "C"s in the sample sequence is replaced by "Y"s. Despite achieving high
genomic coverage, high methylation rate error can be achieved. In the three-character
algorithm alignment method, all "C"s in the reference genome is read as "T". In this case,
genomic coverage decreases (lower capability of mapping). Determining new bioinformatics
algorithms for analyses can increase the effectiveness of the methods.
CONCLUSION
Although some next generation sequencing methods such as DNA methylation analyses,
single-molecule real-time sequencing and Nanopore technologies offer new opportunities for
the identification of new epigenetic markers, they are currently used due to high error rates,
high costs and low volumes. Their use is quite limited compared to second generation
sequencing technologies due to their processing power (lower throughput). Bisulfite based
86
methods are still the gold standard in DNA cytosine methylation studies. BiSeq method is the
choice of the validation studies of WGBS studies for whole genome sequencing studies. BiSeq
studies are also useful for small projects with lower financial supports.
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