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1.  The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing 
PLoS ONE  2010;5(1):e8888.
Background
We recently showed that enzymes of the TET family convert 5-mC to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC is present at high levels in embryonic stem cells and Purkinje neurons. The methylation status of cytosines is typically assessed by reaction with sodium bisulfite followed by PCR amplification. Reaction with sodium bisulfite promotes cytosine deamination, whereas 5-methylcytosine (5-mC) reacts poorly with bisulfite and is resistant to deamination. Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing.
Methodology/Principal Findings
We used synthetic oligonucleotides with different distributions of cytosine as templates for generation of DNAs containing C, 5-mC and 5-hmC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting cytosine deamination. The extent of conversion of 5-hmC to CMS was estimated to be 99.7%. Sequencing of PCR products showed that neither 5-mC nor 5-hmC undergo C-to-T transitions after bisulfite treatment, confirming that these two modified cytosine species are indistinguishable by the bisulfite technique. DNA in which CMS constituted a large fraction of all bases (28/201) was much less efficiently amplified than DNA in which those bases were 5-mC or uracil (the latter produced by cytosine deamination). Using a series of primer extension experiments, we traced the inefficient amplification of CMS-containing DNA to stalling of Taq polymerase at sites of CMS modification, especially when two CMS bases were either adjacent to one another or separated by 1–2 nucleotides.
Conclusions
We have confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-mC and 5-hmC. Moreover, we show that CMS, the product of bisulfite conversion of 5-hmC, tends to stall DNA polymerases during PCR, suggesting that densely hydroxymethylated regions of DNA may be underrepresented in quantitative methylation analyses.
doi:10.1371/journal.pone.0008888
PMCID: PMC2811190  PMID: 20126651
2.  Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine 
PLoS ONE  2014;9(4):e93933.
DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.
doi:10.1371/journal.pone.0093933
PMCID: PMC3974851  PMID: 24699908
3.  Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis — A personal account 
Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called ‘bisulfite genomic sequencing’ was invented by these researchers in 1992. This review describes the author’s reflection of the discovery of bisulfite reactions with pyrimidine bases. The author’s recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.
doi:10.2183/pjab/84.321
PMCID: PMC3722019  PMID: 18941305
bisulfite; 5-methylcytosine; deamination of cytosine; concentration dependency; chemical modification
4.  Kismeth: Analyzer of plant methylation states through bisulfite sequencing 
BMC Bioinformatics  2008;9:371.
Background
There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH).
Results
Kismeth is a web-based tool for bisulfite sequencing analysis. Kismeth was designed to be used with plants, since it considers potential cytosine methylation in any sequence context (CG, CHG, and CHH). It provides a tool for the design of bisulfite primers as well as several tools for the analysis of the bisulfite sequencing results. Kismeth is not limited to data from plants, as it can be used with data from any species.
Conclusion
Kismeth simplifies bisulfite sequencing analysis. It is the only publicly available tool for the design of bisulfite primers for plants, and one of the few tools for the analysis of methylation patterns in plants. It facilitates analysis at both global and local scales, demonstrated in the examples cited in the text, allowing dissection of the genetic pathways involved in DNA methylation. Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.
doi:10.1186/1471-2105-9-371
PMCID: PMC2553349  PMID: 18786255
5.  Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR 
Nucleic Acids Research  2007;35(9):2893-2903.
Many methods for the detection of genomic DNA methylation states have appeared. Currently, nearly all such methods employ bisulfite-mediated deamination of denatured DNA. While this treatment effectively deaminates cytosines to uracils, leaving most 5-methylcytosines intact, it also introduces abasic sites that generate a significant number of single-strand breaks in DNA. We have investigated the interplay of these two processes in order to determine their relative effects on the methylation-sensitive QPCR method. The extent of cleavage of the input DNA is significant and appears to be an increasing function of DNA concentration. Even so, the results suggest that only ∼10% of a 62-nt target will be lost due to degradation and targets up to 131 nt will suffer only a 20% loss. More significant losses were found to occur during the subsequent removal of bisulfite and desulfonation steps that appear to be the result of size selectivity associated with matrix binding and elution required prior to QPCR in the most commonly used protocols. For biospecimens yielding <1 μg of DNA, these findings suggest that bisulfite treatment, in current implementations of MS-QPCR, result in low recoveries that preclude reliable analysis of DNA methylation patterns regardless of target size.
doi:10.1093/nar/gkm055
PMCID: PMC1888819  PMID: 17439964
6.  Bisulfite genomic sequencing: systematic investigation of critical experimental parameters 
Nucleic Acids Research  2001;29(13):e65.
Bisulfite genomic sequencing is the method of choice for the generation of methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil by treatment with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not react with bisulfite and can therefore be distinguished. In order to investigate the potential for optimization of the method and to determine the critical experimental parameters, we determined the influence of incubation time and incubation temperature on the deamination efficiency and measured the degree of DNA degradation during the bisulfite treatment. We found that maximum conversion rates of cytosine occurred at 55°C (4–18 h) and 95°C (1 h). Under these conditions at least 84–96% of the DNA is degraded. To study the impact of primer selection, homologous DNA templates were constructed possessing cytosine-containing and cytosine-free primer binding sites, respectively. The recognition rates for cytosine (≥97%) and 5-methylcytosine (≥94%) were found to be identical for both templates.
PMCID: PMC55789  PMID: 11433041
7.  MethylExtract: High-Quality methylation maps and SNV calling from whole genome bisulfite sequencing data 
F1000Research  2013;2:217.
Whole genome methylation profiling at a single cytosine resolution is now feasible due to the advent of high-throughput sequencing techniques together with bisulfite treatment of the DNA. To obtain the methylation value of each individual cytosine, the bisulfite-treated sequence reads are first aligned to a reference genome, and then the profiling of the methylation levels is done from the alignments. A huge effort has been made to quickly and correctly align the reads and many different algorithms and programs to do this have been created. However, the second step is just as crucial and non-trivial, but much less attention has been paid to the final inference of the methylation states. Important error sources do exist, such as sequencing errors, bisulfite failure, clonal reads, and single nucleotide variants.
We developed MethylExtract, a user friendly tool to: i) generate high quality, whole genome methylation maps and ii) detect sequence variation within the same sample preparation. The program is implemented into a single script and takes into account all major error sources. MethylExtract detects variation (SNVs – Single Nucleotide Variants) in a similar way to VarScan, a very sensitive method extensively used in SNV and genotype calling based on non-bisulfite-treated reads. The usefulness of MethylExtract is shown by means of extensive benchmarking based on artificial bisulfite-treated reads and a comparison to a recently published method, called Bis-SNP.
MethylExtract is able to detect SNVs within High-Throughput Sequencing experiments of bisulfite treated DNA at the same time as it generates high quality methylation maps. This simultaneous detection of DNA methylation and sequence variation is crucial for many downstream analyses, for example when deciphering the impact of SNVs on differential methylation. An exclusive feature of MethylExtract, in comparison with existing software, is the possibility to assess the bisulfite failure in a statistical way. The source code, tutorial and artificial bisulfite datasets are available at http://bioinfo2.ugr.es/MethylExtract/ and http://sourceforge.net/projects/methylextract/, and also permanently accessible from 10.5281/zenodo.7144.
doi:10.12688/f1000research.2-217.v1
PMCID: PMC3938178  PMID: 24627790
8.  MethylExtract: High-Quality methylation maps and SNV calling from whole genome bisulfite sequencing data 
F1000Research  2014;2:217.
Whole genome methylation profiling at a single cytosine resolution is now feasible due to the advent of high-throughput sequencing techniques together with bisulfite treatment of the DNA. To obtain the methylation value of each individual cytosine, the bisulfite-treated sequence reads are first aligned to a reference genome, and then the profiling of the methylation levels is done from the alignments. A huge effort has been made to quickly and correctly align the reads and many different algorithms and programs to do this have been created. However, the second step is just as crucial and non-trivial, but much less attention has been paid to the final inference of the methylation states. Important error sources do exist, such as sequencing errors, bisulfite failure, clonal reads, and single nucleotide variants.
We developed MethylExtract, a user friendly tool to: i) generate high quality, whole genome methylation maps and ii) detect sequence variation within the same sample preparation. The program is implemented into a single script and takes into account all major error sources. MethylExtract detects variation (SNVs – Single Nucleotide Variants) in a similar way to VarScan, a very sensitive method extensively used in SNV and genotype calling based on non-bisulfite-treated reads. The usefulness of MethylExtract is shown by means of extensive benchmarking based on artificial bisulfite-treated reads and a comparison to a recently published method, called Bis-SNP.
MethylExtract is able to detect SNVs within High-Throughput Sequencing experiments of bisulfite treated DNA at the same time as it generates high quality methylation maps. This simultaneous detection of DNA methylation and sequence variation is crucial for many downstream analyses, for example when deciphering the impact of SNVs on differential methylation. An exclusive feature of MethylExtract, in comparison with existing software, is the possibility to assess the bisulfite failure in a statistical way. The source code, tutorial and artificial bisulfite datasets are available at http://bioinfo2.ugr.es/MethylExtract/ and http://sourceforge.net/projects/methylextract/, and also permanently accessible from 10.5281/zenodo.7144.
doi:10.12688/f1000research.2-217.v2
PMCID: PMC3938178  PMID: 24627790
9.  Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies 
Nucleic Acids Research  2008;36(22):e150.
Bisulfite treatment can be used to ascertain the methylation states of individual cytosines in DNA. Ideally, bisulfite treatment deaminates unmethylated cytosines to uracils, and leaves 5-methylcytosines unchanged. Two types of bisulfite-conversion error occur: inappropriate conversion of 5-methylcytosine to thymine, and failure to convert unmethylated cytosine to uracil. Conventional bisulfite treatment requires hours of exposure to low-molarity, low-temperature bisulfite (‘LowMT’) and, sometimes, thermal denaturation. An alternate, high-molarity, high-temperature (‘HighMT’) protocol has been reported to accelerate conversion and to reduce inappropriate conversion. We used molecular encoding to obtain validated, individual-molecule data on failed- and inappropriate-conversion frequencies for LowMT and HighMT treatments of both single-stranded and hairpin-linked oligonucleotides. After accounting for bisulfite-independent error, we found that: (i) inappropriate-conversion events accrue predominantly on molecules exposed to bisulfite after they have attained complete or near-complete conversion; (ii) the HighMT treatment is preferable because it yields greater homogeneity among sites and among molecules in conversion rates, and thus yields more reliable data; (iii) different durations of bisulfite treatment will yield data appropriate to address different experimental questions; and (iv) conversion errors can be used to assess the validity of methylation data collected without the benefit of molecular encoding.
doi:10.1093/nar/gkn691
PMCID: PMC2602783  PMID: 18984622
10.  Bisulfite Sequencing of DNA 
Exact positions of 5-methylcytosine (m5C) on a single strand of DNA can be determined by bisulfite genomic sequencing (BGS). Treatment with bisulfite ion preferentially deaminates unmethylated cytosines, which then convert to uracil upon desulfonation. Amplifying regions of interest from deaminated DNA and sequencing products cloned from amplicons permits determination of methylation at single nucleotide resolution along single DNA molecules, which is not possible with other methylation analysis techniques. This unit describes a BGS technique suitable for most DNA sources, including formaldehyde-fixed tissue. Considerations for experimental design and common sources of error are discussed.
doi:10.1002/0471142727.mb0709s91
PMCID: PMC3214597  PMID: 20583099
11.  GPU-BSM: A GPU-Based Tool to Map Bisulfite-Treated Reads 
PLoS ONE  2014;9(5):e97277.
Cytosine DNA methylation is an epigenetic mark implicated in several biological processes. Bisulfite treatment of DNA is acknowledged as the gold standard technique to study methylation. This technique introduces changes in the genomic DNA by converting cytosines to uracils while 5-methylcytosines remain nonreactive. During PCR amplification 5-methylcytosines are amplified as cytosine, whereas uracils and thymines as thymine. To detect the methylation levels, reads treated with the bisulfite must be aligned against a reference genome. Mapping these reads to a reference genome represents a significant computational challenge mainly due to the increased search space and the loss of information introduced by the treatment. To deal with this computational challenge we devised GPU-BSM, a tool based on modern Graphics Processing Units. Graphics Processing Units are hardware accelerators that are increasingly being used successfully to accelerate general-purpose scientific applications. GPU-BSM is a tool able to map bisulfite-treated reads from whole genome bisulfite sequencing and reduced representation bisulfite sequencing, and to estimate methylation levels, with the goal of detecting methylation. Due to the massive parallelization obtained by exploiting graphics cards, GPU-BSM aligns bisulfite-treated reads faster than other cutting-edge solutions, while outperforming most of them in terms of unique mapped reads.
doi:10.1371/journal.pone.0097277
PMCID: PMC4026317  PMID: 24842718
12.  A new method for accurate assessment of DNA quality after bisulfite treatment 
Nucleic Acids Research  2007;35(5):e29.
The covalent addition of methylgroups to cytosine has become the most intensively researched epigenetic DNA marker. The vast majority of technologies used for DNA methylation analysis rely on a chemical reaction, the so-called ‘bisulfite treatment’, which introduces methylation-dependent sequence changes through selective chemical conversion of non-methylated cytosine to uracil. After treatment, all non-methylated cytosine bases are converted to uracil but all methylated cytosine bases remain cytosine. These methylation dependent C-to-T changes can subsequently be studied using conventional DNA analysis technologies.
The bisulfite conversion protocol is susceptible to processing errors, and small deviation from the protocol can result in failure of the treatment. Several attempts have been made to simplify the procedure and increase its robustness. Although significant achievements in this area have been made, bisulfite treatment remains the main source of process variability in the analysis of DNA methylation. This variability in particular impairs assays, which strive for the quantitative assessment of DNA methylation. Here we present basic mathematical considerations, which should be taken into account when analyzing DNA methylation. We also introduce a PCR-based assay, which allows ab initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment.
doi:10.1093/nar/gkl1134
PMCID: PMC1865059  PMID: 17259213
13.  A Bayesian Framework to Identify Methylcytosines from High-Throughput Bisulfite Sequencing Data 
PLoS Computational Biology  2014;10(9):e1003853.
High-throughput bisulfite sequencing technologies have provided a comprehensive and well-fitted way to investigate DNA methylation at single-base resolution. However, there are substantial bioinformatic challenges to distinguish precisely methylcytosines from unconverted cytosines based on bisulfite sequencing data. The challenges arise, at least in part, from cell heterozygosis caused by multicellular sequencing and the still limited number of statistical methods that are available for methylcytosine calling based on bisulfite sequencing data. Here, we present an algorithm, termed Bycom, a new Bayesian model that can perform methylcytosine calling with high accuracy. Bycom considers cell heterozygosis along with sequencing errors and bisulfite conversion efficiency to improve calling accuracy. Bycom performance was compared with the performance of Lister, the method most widely used to identify methylcytosines from bisulfite sequencing data. The results showed that the performance of Bycom was better than that of Lister for data with high methylation levels. Bycom also showed higher sensitivity and specificity for low methylation level samples (<1%) than Lister. A validation experiment based on reduced representation bisulfite sequencing data suggested that Bycom had a false positive rate of about 4% while maintaining an accuracy of close to 94%. This study demonstrated that Bycom had a low false calling rate at any methylation level and accurate methylcytosine calling at high methylation levels. Bycom will contribute significantly to studies aimed at recalibrating the methylation level of genomic regions based on the presence of methylcytosines.
Author Summary
High-throughput bisulfite sequencing (BS-seq) has advanced tremendously the study of DNA methylation and the determination of methylcytosines at single-base resolution. In BS-seq data analysis, sequencing errors, incomplete bisulfite conversion, and cell heterozygosis affect the accuracy of methylcytosine detection in quite a major way. Simple filtering methods using predefined thresholds have proved to have extremely low efficiency. The commonly used Lister uses binomial distribution to overcome the impacts of non-conversion rate and sequencing errors, but the impact of the cell heterozygosis is not considered. Here, we present Bycom, a novel algorithm based on the Bayesian inference model. To improve the accuracy of methylcytosine calling, Bycom considers sequencing errors, non-conversion rate, and cell heterozygosis integratively to identify methylcytosines from BS-seq data. We evaluated the performance of Bycom using different kinds of BS-seq data. Our results demonstrated that Bycom identified methylcytosines more accurately than Lister, especially in BS-seq data with extremely low genome-wide methylation levels.
doi:10.1371/journal.pcbi.1003853
PMCID: PMC4177668  PMID: 25255082
14.  DNA Methylation: Bisulphite Modification and Analysis 
Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.
doi:10.3791/3170
PMCID: PMC3227193  PMID: 22042230
15.  DNA Methylation: A Timeline of Methods and Applications 
DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. An epigenetic modification associated with gene regulation, DNA methylation is of paramount importance to biological health and disease. Recently, the quest to unravel the Human Epigenome commenced, calling for a modernization of previous DNA methylation profiling techniques. Here, we describe the major developments in the methodologies used over the past three decades to examine the elusive epigenome (or methylome). The earliest techniques were based on the separation of methylated and unmethylated cytosines via chromatography. The following years would see molecular techniques being employed to indirectly examine DNA methylation levels at both a genome-wide and locus-specific context, notably immunoprecipitation via anti-5′methylcytosine and selective digestion with methylation-sensitive restriction endonucleases. With the advent of sodium bisulfite treatment of DNA, a deamination reaction that converts cytosine to uracil only when unmethylated, the epigenetic modification can now be identified in the same manner as a DNA base-pair change. More recently, these three techniques have been applied to more technically advanced systems such as DNA microarrays and next-generation sequencing platforms, bringing us closer to unveiling a complete human epigenetic profile.
doi:10.3389/fgene.2011.00074
PMCID: PMC3268627  PMID: 22303369
DNA; methylation; bisulfite; sequencing; methods
16.  Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). 
Nucleic Acids Research  1997;25(12):2529-2531.
We have developed a rapid quantitative method (Ms-SNuPE) for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single nucleotide primer extension. Genomic DNA was first reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence was then performed using PCR primers specific for bisulfite-converted DNA and the resulting product isolated and used as a template for methylation analysis at the CpG site(s) of interest. This methylation-sensitive technique has several advantages over existing methods used for detection of methylation changes because small amounts of DNA can be analyzed including microdissected pathology sections and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.
PMCID: PMC146734  PMID: 9171109
17.  MethTools—a toolbox to visualize and analyze DNA methylation data 
Nucleic Acids Research  2000;28(5):1053-1058.
The Bisulfite Genomic Sequencing technique has found wide acceptance for the generation of DNA-methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil (and subsequent conversion to thymine via PCR), whereas 5-methylcytosine residues remain unchanged. Methylation maps are created by the comparison of bisulfite converted sequences with the untreated genomic sequence. ‘MethTools’ is a collection of software tools that replaces the time-consuming manual comparison process, generates graphical outputs of methylation patterns and methylation density, estimates the systematic error of the experiment and searches for conserved methylated nucleotide patterns. The programs are written in Perl 5 and C, and the source code can be downloaded. All tools run independently but the programs are interfaced. Thus, a script can perform the entire analysis procedure automatically. In addition, a web-based remote analysis service is offered. Both the source code and the remote analysis are available at http://genome.imb-jena.de/methtools/
PMCID: PMC102603  PMID: 10666443
18.  CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene. 
Molecular and Cellular Biology  1994;14(12):7975-7983.
Inactive-X-chromosome genes in mammalian females have methylated CpG islands. We have questioned whether there are variable levels of cytosine methylation at different CpG sites within the island that might indicate the presence of primary sites of methylation which may be critical for the maintenance of gene repression and candidate sites for the initiation of inactivation. To address these questions, we have analyzed the methylation patterns of 32 CpG sites of the X-linked hypoxanthine phosphoribosyltransferase (Hprt) gene on the active and inactive X chromosomes of mouse tissues and cell lines, using genomic sequencing of bisulfite-treated genomic DNA. Cytosine is deaminated by bisulfite, but methylcytosine is not affected. Cell lines that were heterozygous for the Hprt deletion mutation (Hprtb-m3) and a functional Hprt allele were selected with 6-thioguanine. The resulting cell populations uniformly carry the intact Hprt allele on the inactive X chromosome. The methylation of these CpG sites was determined either by the direct sequence analysis of bisulfite-treated and amplified DNA or by the sequence analysis of clones derived from the amplified DNA. No CpG methylation was detected on the active Hprt genes from either males or the active X chromosome of females. On average, 22 CpGs were methylated in the other 50% of female DNA, and the level of methylation at individual sites varied from 42 to 100%. Analysis of the inactive Hprt gene in two cell lines showed that averages of 14 and 18 CpGs were methylated and that the frequency of methylation at 32 individual sites ranged from 3 to 100%. The highest frequency of methylation in cell lines coincided with the sequences flanking transcription initiation sites. These results suggest that methylation patterns are heterogeneous within a tissue and even in clonal cell populations and that specific subsets of CpG sites sustain high methylation frequencies which may be critical for the maintenance of X-chromosome inactivation. The bisulfite method identified which CpG sites were methylated on the inactive X chromosome, and it provided a quantitative estimate of the frequency of methylation of these sites in genomic DNA.
Images
PMCID: PMC359336  PMID: 7969137
19.  Pyrosequencing Evaluation of Widely Available Bisulfite Conversion Methods: Considerations for Application 
Medical epigenetics  2014;2(1):28-36.
Introduction
Bisulfite treatment of DNA introduces methylation-dependent sequence changes through selective chemical conversion of nonmethylated cytosine to uracil and serves as pretreatment step for the majority of DNA methylation analysis methods.
Methods
We have evaluated the conversion performance of five of the most commonly used bisulfite treatment kits [MethylDetector (Active Motif), Epitect+ (Qiagen), Zymo Methylation, Zymo Gold and Zymo Lightning (all from Zymo Research)] by pyrosequencing four different regions with variable methylation levels, including: a repetitive element (ALUSX), a gene with low levels of methylation (IL6ST), an imprinted gene expected to be approximately 50% methylated (IGF2), and a fully methylated gene (ST3GAL2). In addition, we have studied the influence of duration (3 vs. 16 h) and type (fixed temperature vs. cycling program) of incubation protocol on the conversion efficiency of each evaluated kit.
Results
All kits produced similar conversion rates of ALUSX, IGF2 and ST3GAL2, while the conversion of the low methylated IL6ST gene was variable between kits. The Zymo kits were highly consistent in their performance even when different protocols of incubation were applied, generating full conversion at the low methylated gene IL6; this was not true for the MethylDetector and Epitect+ kits. However, long-cycling incubation could produce similar conversion rates for the same locus in combination with Active Motif and Qiagen kits.
Conclusions
The selection of a long-cycling protocol during conversion permits standardization of protocols, improving the reproducibility of methylation estimates across laboratories for gene-specific, genome-wide and bisulfite-based sequencing analyses.
doi:10.1159/000358882
PMCID: PMC4058339  PMID: 24944560
DNA methylation; Bisulfite treatment; Pyrosequencing Abstract
20.  MethyQA: a pipeline for bisulfite-treated methylation sequencing quality assessment 
BMC Bioinformatics  2013;14:259.
Background
DNA methylation is an epigenetic event that adds a methyl-group to the 5’ cytosine. This epigenetic modification can significantly affect gene expression in both normal and diseased cells. Hence, it is important to study methylation signals at the single cytosine site level, which is now possible utilizing bisulfite conversion technique (i.e., converting unmethylated Cs to Us and then to Ts after PCR amplification) and next generation sequencing (NGS) technologies. Despite the advances of NGS technologies, certain quality issues remain. Some of the more prevalent quality issues involve low per-base sequencing quality at the 3’ end, PCR amplification bias, and bisulfite conversion rates. Therefore, it is important to conduct quality assessment before downstream analysis. To the best of our knowledge, no existing software packages can generally assess the quality of methylation sequencing data generated based on different bisulfite-treated protocols.
Results
To conduct the quality assessment of bisulfite methylation sequencing data, we have developed a pipeline named MethyQA. MethyQA combines currently available open-source software packages with our own custom programs written in Perl and R. The pipeline can provide quality assessment results for tens of millions of reads in under an hour. The novelty of our pipeline lies in its examination of bisulfite conversion rates and of the DNA sequence structure of regions that have different conversion rates or coverage.
Conclusions
MethyQA is a new software package that provides users with a unique insight into the methylation sequencing data they are researching. It allows the users to determine the quality of their data and better prepares them to address the research questions that lie ahead. Due to the speed and efficiency at which MethyQA operates, it will become an important tool for studies dealing with bisulfite methylation sequencing data.
doi:10.1186/1471-2105-14-259
PMCID: PMC3765750  PMID: 23968174
DNA methylation; Next generation sequencing; Alignment; BRAT; Quality assessment
21.  Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding 
PLoS ONE  2010;5(2):e9320.
Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD™ System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD™ bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.
doi:10.1371/journal.pone.0009320
PMCID: PMC2825269  PMID: 20179767
22.  Increased Sensitivity in Whole-Genome Bisulfite Sequencing (Wgbs): A Novel “Post-bisulfite Conversion” Library Construction Method from Sub-nanogram DNA Inputs 
Genome-wide analysis of 5-methylcytosines is possible with whole-genome bisulfite sequencing (WGBS), where unmethylated cytosine residues are converted to uracil. However, a major challenge in WGBS is the degradation of DNA that occurs during bisulfite conversion under conditions required for complete conversion. Typically, ∼90% of input DNA is degraded and thus, is especially problematic with limited starting amounts of DNA. Additionally, regions that are rich in unmethylated cytosines are more sensitive to strand breaks. As a consequence, a majority of DNA fragments contained in di-tagged NGS DNA libraries treated with bisulfite “post-library construction” can be rendered inactive due to strand breaks in the DNA sequence flanked by the adapter sequences. These mono-tagged templates are then excluded during library enrichment resulting in incomplete coverage and bias when performing whole genome bisulfite sequencing.
Here, we describe a novel “post-bisulfite conversion” library construction method for preparing NGS libraries from genomic DNA prior to the addition of one or both adapters. This “post-bisulfite conversion” library construction method uses the resulting untagged or mono-tagged single-stranded DNA as template for the subsequent addition of adapter sequences required for NGS. Thus, single-stranded DNA fragments independent of size and position of strand breaks remain as viable templates for library construction, eliminating the loss of fragments and the selection bias associated with a “post-library construction” bisulfite conversion strategy. This novel “post-bisulfite conversion” library construction method exhibits high diversity, increased efficiency and sensitivity (500 picograms human genomic DNA input), and improved coverage required for WGBS.
PMCID: PMC3635258
23.  A method for cloning and sequencing long palindromic DNA junctions 
Nucleic Acids Research  2004;32(19):e155.
DNA sequences containing long adjacent inverted repeats (palindromes) are inherently unstable and are associated with many types of chromosomal rearrangements. The instability associated with palindromic sequences also creates difficulties in their molecular analysis: long palindromes (>250 bp/arm) are highly unstable in Escherichia coli, and cannot be directly PCR amplified or sequenced due to their propensity to form intra-strand hairpins. Here, we show that DNA molecules containing long palindromes (>900 bp/arm) can be transformed and stably maintained in Saccharomyces cerevisiae cells lacking a functional SAE2 gene. Treatment of the palindrome-containing DNA with sodium bisulfite at high temperature results in deamination of cytosine, converting it to uracil and thus reducing the propensity to form intra-strand hairpins. The bisulfite-treated DNA can then be PCR amplified, cloned and sequenced, allowing determination of the nucleotide sequence of the junctions. Our data demonstrates that long palindromes with either no spacer (perfect) or a 2 bp spacer can be stably maintained, recovered and sequenced from sae2Δ yeast cells. Since DNA sequences from mammalian cells can be gap repaired by their co-transformation into yeast cells with an appropriate vector, the methods described in this manuscript should provide some of the necessary tools to isolate and characterize palindromic junctions from mammalian cells.
doi:10.1093/nar/gnh143
PMCID: PMC528819  PMID: 15534362
24.  An AP Endonuclease Functions in Active DNA Dimethylation and Gene Imprinting in Arabidopsis 
PLoS Genetics  2015;11(1):e1004905.
Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/−zdp−/− mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.
Author Summary
DNA cytosine methylation (5-methylcytosine, 5-meC) is an important epigenetic mark, and methylation patterns are coordinately controlled by methylation and demethylation reactions during development and reproduction. In plants, REPRESSOR OF SILENCING (ROS1) is one of the well characterized 5-meC DNA glycosylases that initiate active DNA demethylation by 5-meC excision. Our previous work showed that a 3′-DNA phosphatase, ZDP, functions downstream of ROS1 during active DNA demethylation in Arabidopsis. Here we found that the apurinic/apyrimidinic endonuclease APE1L functions downstream of ROS1 in a ZDP-independent branch of the active DNA demethylation pathway in Arabidopsis. In plants, gene imprinting requires the 5-meC DNA glycosylase Demeter (DME) that has been proposed to initiate a base excision repair pathway for active DNA demethylation in the central cell in female gametophyte. However, besides DME, no other base excision repair enzymes have been found to be important for gene imprinting. Our results show that APE1L and ZDP act jointly downstream of DME to regulate gene imprinting in plants, and suggest that DME-initiated active DNA demethylation in the central cell and endosperm uses both APE- and ZDP-dependent mechanisms.
doi:10.1371/journal.pgen.1004905
PMCID: PMC4287435  PMID: 25569774
25.  Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. 
Nucleic Acids Research  1980;8(20):4777-4790.
Sodium bisulfite is a mutagen which can specifically deaminate more than 96% of the cytosine residues in single-stranded DNA via formation of a 5,6-dihydrocytosine-6-sulfonate intermediate. Under the same reaction conditions, only 2-3% of the 5-methylcytosine (m5Cyt) residues in single-stranded XP-12 DNA, which has 34 mole% m5Cyt, was converted to thymine (Thy) residues. In contrast, at the deoxynucleoside and free base levels, the same treatment with bisulfite and then alkali converted 51% and > 95%, respectively, of the m5Cyt to the corresponding Thy derivatives. However, the rate of reaction of m5Cyt and its deoxyribonucleoside was much slower than that of the analogous quantitative conversion of cytosine or deoxycytidine to uracil or deoxyuridine, respectively. The much lower reactivity of m5Cyt and its derivatives compared to that of the unmethylated analogs is primarily due to a decrease in the rate of formation of the sulfonate adduct.
PMCID: PMC324387  PMID: 7443525

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