BSMAP: whole genome bisulfite sequence MAPping program

Background  

Bisulfite sequencing is a powerful technique to study DNA cytosine methylation. Bisulfite treatment followed by PCR amplification specifically converts unmethylated cytosines to thymine. Coupled with next generation sequencing technology, it is able to detect the methylation status of every cytosine in the genome. However, mapping high-throughput bisulfite reads to the reference genome remains a great challenge due to the increased searching space, reduced complexity of bisulfite sequence, asymmetric cytosine to thymine alignments, and multiple CpG heterogeneous methylation.     

The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing

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.     

High-Speed Conversion of Cytosine to Uracil in Bisulfite Genomic Sequencing Analysis of DNA Methylation

Bisulfite genomic sequencing is a widely used technique foranalyzing cytosine-methylation of DNA. By treating DNA withbisulfite, cytosine residues are deaminated to uracil, whileleaving 5-methylcytosine largely intact. Subsequent PCR andnucleotide sequence analysis permit unequivocal determinationof the methylation status at cytosine residues. A major caveatassociated with the currently practiced procedure is that ittakes 16–20 hr for completion of the conversion of cytosineto uracil. Here we report that a complete deamination of cytosineto uracil can be achieved in shorter periods by using a highlyconcentrated bisulfite solution at an elevated temperature.Time course experiments demonstrated that treating DNA with9 M bisulfite for 20 min at 90°C or 40 min at 70°C allcytosine residues in the DNA were converted to uracil. Underthese conditions, the majority of 5-methylcytosines remainedintact. When a high molecular weight DNA derived from a cellline (containing a number of genes whose methylation statuswas known) was treated with bisulfite under the above conditionsand amplified and sequenced, the results obtained were consistentwith those reported in the literature. Although some degradationof DNA occurred during this process, the amount of treated DNArequired for the amplification was nearly equal to that requiredfor the conventional bisulfite genomic sequencing procedure.The increased speed of DNA methylation analysis with this novelprocedure is expected to advance various aspects of DNA sciences.     

A new method for accurate assessment of DNA quality after bisulfite treatment

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.

     

Directional telomeric silencing and lack of canonical B1 elements in two silencer Autonomously Replicating Sequences in S. cerevisiae

Background

Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amounts of cell-free DNA in plasma, analytical sensitivity is of utmost importance. The vast majority of currently available methods for analysing DNA methylation are based on bisulfite-mediated deamination of cytosine. Cytosine is rapidly converted to uracil during bisulfite treatment, whereas 5-methylcytosine is only slowly converted. Hence, bisulfite treatment converts an epigenetic modification into a difference in sequence, amenable to analysis either by sequencing or PCR based methods. However, the recovery of bisulfite-converted DNA is very poor.

Results

Here we introduce an alternative method for the crucial steps of bisulfite treatment with high recovery. The method is based on an accelerated deamination step and alkaline desulfonation in combination with magnetic silica purification of DNA, allowing preparation of deaminated DNA from patient samples in less than 2 hours.

Conclusions

The method presented here allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma.     

The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR,Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR

Background  

Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples.     

Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.     

The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research

Methylation at position 5 of cytosine in DNA is an important event in epigenetic changes of cells, the methylation being linked to the control of gene functions. The DNA methylation can be analyzed by bisulfite genomic sequencing, and a large body of data have now been accumulated, based on which causation of diseases, for example cancer, and many other manifestations of cellular activities have been discussed intensively. This article gives an extensive account of the chemical aspects of bisulfite modification of cytosine and 5-methylcytosine in DNA. Various factors that affect the action of bisulfite are discussed, and a recent progress from our laboratory is explained. Conventional procedures for the bisulfite treatment consist incubation of single-stranded DNA with sodium bisulfite under acidic conditions. This treatment converts cytosine into uracil, but 5-methylcytosine remains unchanged. Amplification by polymerase chain reaction (PCR) of the bisulfite-treated DNA followed by sequencing can result in revealing the positions of 5-methylcytosine in the gene. We have discovered that the whole procedure can be significantly speeded up by the use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. Another recent finding is that urea, which has been often added to the reaction mixture with the purpose of facilitating the bisulfite-mediated deamination of cytosine in DNA, may not work as anticipated: we have observed that urea does not show such promoting actions in our treatments of DNA. A laboratory protocol for quantifying bisulfite, suitably simple for routine practice to ensure valid experiments, is described.     

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.     

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax

Background

Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells.

Results

Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax. These DNA modifications occur only during nuclear development and programmed genome rearrangement. We detect methylcytosine and hydroxymethylcytosine directly by high-resolution nano-flow UPLC mass spectrometry, and indirectly by immunofluorescence, methyl-DNA immunoprecipitation and bisulfite sequencing. We describe these modifications in three classes of eliminated DNA: germline-limited transposons and satellite repeats, aberrant DNA rearrangements, and DNA from the parental genome undergoing degradation. Methylation and hydroxymethylation generally occur on the same sequence elements, modifying cytosines in all sequence contexts. We show that the DNA methyltransferase-inhibiting drugs azacitidine and decitabine induce demethylation of both somatic and germline sequence elements during genome rearrangements, with consequent elevated levels of germline-limited repetitive elements in exconjugant cells.

Conclusions

These data strongly support a functional link between cytosine DNA methylation/hydroxymethylation and DNA elimination. We identify a motif strongly enriched in methylated/hydroxymethylated regions, and we propose that this motif recruits DNA modification machinery to specific chromosomes in the parental macronucleus. No recognizable methyltransferase enzyme has yet been described in O. trifallax, raising the possibility that it might employ a novel cytosine methylation machinery to mark DNA sequences for elimination during genome rearrangements.     

Bisulfite genomic sequencing: systematic investigation of critical experimental parameters

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.     

Microsoft Word macro for analysis of cytosine methylation by the bisulfite deamination reaction

Cytosine methylation at CpG dinucleotides is an important control mechanism in development, differentiation, and neoplasia. Bisulfite genomic sequencing and its modifications have been developed to examine methylation at these CpG dinucleotides. To use these methods, one has to (i) manually convert the sequence to that produced by bisulfite conversion and PCR amplification, taking into account that cytosine residues at CpG dinucleotides may or may not be converted depending on their methylation status, (ii) identify relevant restriction sites that may be used for methylation analysis, and (iii) conduct similar steps with the other DNA strand since the two strands of DNA are no longer complementary after bisulfite conversion. To automate these steps, we have developed a macro that can be used with Microsoft Word. This macro (i) converts genomic sequence to modified sequence that would result after bisulfite treatment facilitating primer design for bisulfite genomic sequencing and methylation-sensitive PCR assay and (ii) identifies restriction sites that are preserved in bisulfite-converted and PCR-amplified product only if cytosine residues at relevant CpG dinucleotides are methylated (and thereby not converted to uracil) in the genomic DNA.     

A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor. We believe that this method may easily be adapted for analyzing other genes and provide guidelines for selecting the most appropriate MRED restriction enzymes.     

Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates

MOTIVATION: Methylation of cytosines in DNA plays an important role in the regulation of gene expression, and the analysis of methylation patterns is fundamental for the understanding of cell differentiation, aging processes, diseases and cancer development. Such analysis has been limited, because technologies for detailed and efficient high-throughput studies have not been available. We have developed a novel quantitative methylation analysis algorithm and workflow based on direct DNA sequencing of PCR products from bisulfite-treated DNA with high-throughput sequencing machines. This technology is a prerequisite for success of the Human Epigenome Project, the first large genome-wide sequencing study for DNA methylation in many different tissues. Methylation in tissue samples which are compositions of different cells is a quantitative information represented by cytosine/thymine proportions after bisulfite conversion of unmethylated cytosines to uracil and PCR. Calculation of quantitative methylation information from base proportions represented by different dye signals in four-dye sequencing trace files needs a specific algorithm handling imbalanced and overscaled signals, incomplete conversion, quality problems and basecaller artifacts. RESULTS: The algorithm we developed has several key properties: it analyzes trace files from PCR products of bisulfite-treated DNA sequenced directly on ABI machines; it yields quantitative methylation measurements for individual cytosine positions after alignment with genomic reference sequences, signal normalization and estimation of effectiveness of bisulfite treatment; it works in a fully automated pipeline including data quality monitoring; it is efficient and avoids the usual cost of multiple sequencing runs on subclones to estimate DNA methylation. The power of our new algorithm is demonstrated with data from two test systems based on mixtures with known base compositions and defined methylation. In addition, the applicability is proven by identifying CpGs that are differentially methylated in real tissue samples.     

Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA

In this study, we adapted the well known uracil DNA glycosylase (UNG) carry-over prevention system for PCR, and applied it to the analysis of DNA methylation based on sodium bisulfite conversion. As sodium bisulfite treatment converts unmethylated cytosine bases into uracil residues, bisulfite treated DNA is sensitive to UNG treatment. Therefore, UNG cannot be used for carry-over prevention of PCR using bisulfite treated template DNA, as not only contaminating products of previous PCR, but also the actual template will be degraded. We modified the bisulfite treatment procedure and generated DNA containing sulfonated uracil residues. Surprisingly, and in contrast to uracil, 6-sulfonyl uracil containing DNA (SafeBis DNA) is resistant to UNG. We showed that the new procedure removes up to 10 000 copies of contaminating PCR product in a closed PCR vessel without significant loss of analytical or clinical sensitivity of the DNA methylation analysis.     

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

Background

Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis.

Methods

Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing.

Results

The main features and advantages of this protocol are:

• An optimized method for extracting good quality DNA from FFPE tissues.
• An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue.
• Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing.

Conclusions

We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

     

Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia

Background  

Establishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.     

Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples

Background

DNA methylation plays crucial roles in epigenetic gene regulation in normal development and disease pathogenesis. Efficient and accurate quantification of DNA methylation at single base resolution can greatly advance the knowledge of disease mechanisms and be used to identify potential biomarkers. We developed an improved pipeline based on reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide quantification of DNA methylation at single base resolution. A selection of two restriction enzymes (Taq^αI and MspI) enables a more unbiased coverage of genomic regions of different CpG densities. We further developed a highly automated software package to analyze bisulfite sequencing results from the Solexa GAIIx system.

Results

With two sequencing lanes, we were able to quantify ~1.8 million individual CpG sites at a minimum sequencing depth of 10. Overall, about 76.7% of CpG islands, 54.9% of CpG island shores and 52.2% of core promoters in the human genome were covered with at least 3 CpG sites per region.

Conclusions

With this new pipeline, it is now possible to perform whole-genome DNA methylation analysis at single base resolution for a large number of samples for understanding how DNA methylation and its changes are involved in development, differentiation, and disease pathogenesis.     

CpG_MPs: identification of CpG methylation patterns of genomic regions from high-throughput bisulfite sequencing data

High-throughput bisulfite sequencing is widely used to measure cytosine methylation at single-base resolution in eukaryotes. It permits systems-level analysis of genomic methylation patterns associated with gene expression and chromatin structure. However, methods for large-scale identification of methylation patterns from bisulfite sequencing are lacking. We developed a comprehensive tool, CpG_MPs, for identification and analysis of the methylation patterns of genomic regions from bisulfite sequencing data. CpG_MPs first normalizes bisulfite sequencing reads into methylation level of CpGs. Then it identifies unmethylated and methylated regions using the methylation status of neighboring CpGs by hotspot extension algorithm without knowledge of pre-defined regions. Furthermore, the conservatively and differentially methylated regions across paired or multiple samples (cells or tissues) are identified by combining a combinatorial algorithm with Shannon entropy. CpG_MPs identified large amounts of genomic regions with different methylation patterns across five human bisulfite sequencing data during cellular differentiation. Different sequence features and significantly cell-specific methylation patterns were observed. These potentially functional regions form candidate regions for functional analysis of DNA methylation during cellular differentiation. CpG_MPs is the first user-friendly tool for identifying methylation patterns of genomic regions from bisulfite sequencing data, permitting further investigation of the biological functions of genome-scale methylation patterns.     

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.