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.     

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.     

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.     

Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies

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.     

Microarray-based methylation analysis using dual-color fluorescence hybridization

BACKGROUND: Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple, high-throughput and quantitative methods for methylation detection. METHODS: A high-throughput methylation analysis method has been developed based on microarray and dual-color fluorescence hybridization. The genomic DNA was treated with bisulfite, resulting in conversion of non-methylated cytosine, but not methylated cytosine, into uracil within CpG islands of interest. PCR products of the treated genomic templates were spotted and immobilized onto a poly-l-lysine coated glass slide to fabricate a microarray and then interrogated by hybridization with dual-color probes to determine the methylation status. The hybridized signals were obtained with a scanner and the results were analyzed with the software Genepix Pro 3.0. RESULTS: The methylation status of the CpG islands of IGFBP7 gene has been successfully evaluated by the microarray method for twenty-seven samples. All the investigated samples, including twenty human breast tumor tissues, six corresponding normal human breast tissues and one liver cell line, all CpG sites were found completely methylated. CONCLUSIONS: The microarray technology has been proven to have potential for high-throughput detection of the methylation status for a given gene in multi-genomic samples, which could be a novel approach for rapidly screening DNA methylation marker for early stage cancer diagnosis.     

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.     

Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. Here, predicted changes in restriction enzyme sites following reaction of genomic DNA with bisulfite and amplification of the product by the polymerase chain reaction (PCR) were used to assess the methylation of CpG sites. This procedure differs from conventional DNA methylation analysis by methylation-sensitive restriction enzymes because it does not rely on an absence of cleavage to detect methylated sites, the two strands of DNA produce different restriction enzyme sites and may be differentially analyzed, and closely related sequences may be separately analyzed by using specific PCR primers.     

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.     

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.     

Identification and resolution of artifacts in bisulfite sequencing

Bisulfite sequencing has become the most widely used application to detect 5-methylcytosine (5-MeC) in DNA, and provides a reliable way of detecting any methylated cytosine at single-molecule resolution in any sequence context. The process of bisulfite treatment exploits the different sensitivity of cytosine and 5-MeC to deamination by bisulfite under acidic conditions, in which cytosine undergoes conversion to uracil while 5-MeC remains unreactive. In this article, we address the more commonly encountered experimental artifacts associated with bisulfite sequencing, and provide methods for the detection and elimination of these artifacts. In particular, we focus on conditions that inhibit complete bisulfite-mediated conversion of cytosines in a target sequence, and demonstrate the necessity of complete protein removal from DNA samples prior to bisulfite treatment. We also include a brief summary of the experimental protocol for bisulfite treatment and tips for designing polymerase chain reaction (PCR) primers to amplify from bisulfite-treated DNA.     

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.     

Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform

DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management.     

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.     

Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE).

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.     

Reaction of bisulfite with the 5-hydroxymethyl group in pyrimidines and in phage DNAs.

5-Hydroxymethylcytosine reacted with bisulfite and, instead of undergoing usual deamination process, gave cytosine 5-methylenesulfonate as the product. The conversion was rapid and quantitative, and the optimum pH was 4.5. The product was isolated as crystals and characterized. Cytosine 5-methylenesulfonate was only very slowly deaminated by treatment with bisulfite. 5-Hydroxymethyl-2'-deoxycytidine 5'-phosphate reacted with bisulfite in the same way as 5-hydroxymethylcytosine. Residues of 5-hydroxymethylcytosine in native as well as denatured T2 DNA were convertible to those of cytosine 5-methylenesulfonate by treatment of the DNA with bisulfite. While it is known that the 5-hydroxy-methyl groups of T-even bacteriophage DNA can be enzymatically glucosylated, this observation offers chemical evidence that the 5-hydrozymethyl groups in DNA are situated in such a way that they can readily react with external agents. 5-Hydroxymethyluracil gave uracil 5-methylenesulfonate on treatment with bisulfite. This reaction was much slower than that of 5-hydroxymethylcytosine, and the optimum pH was between 6 and 7.     

Kismeth: Analyzer of plant methylation states through bisulfite sequencing

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).     

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.     

Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA

DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil–DNA–glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms.     

Tritium labelling of nucleic acids accompanied by conversion of cytosine to uracil.

A new method of incorporation of tritium into nucleic acids with an accompanying conversion of cytosine to uracil is proposed. The method is based on the reaction of nucleic acids with bisulfite in the presence of 3H2O. Under certain conditions poly(C) is quantitatively converted to a radioactive poly(U), whereas similar bisulfite treatment of poly(U) does not result in any tritium incorporation. Specificity of the reaction is confirmed by the results of analysis of modified tRNA and rRNA. Incubation of tRNA with bisulfite and 3H2O does not lead to cleavage of the polynucleotide chain. Similar treatment of the denatured DNA results in tritium incorporation into DNA which is accompanied by a conversion of cytosine to uracil. There is virtually no reaction between native DNA and bisulfite. Only certain cytosone residues in yeast tRNAVal/2a interact with bisulfate providing that reaction is carried out under sufficiently mild conditions.