methGraph: A genome visualization tool for PCR-based methylation assays

Abnormalities in DNA methylation of CpG islands that play a role in gene regulation affect gene expression and hence play a role in disease, including cancer. Bisulfite-based DNA methylation analysis methods such as methylation-specific PCR (MSP) and bisulfite sequencing (BiSeq) are most commonly used to study gene-specific DNA methylation. Assessing specificity and visualizing the position of PCR primers in their genomic context is a laborious and tedious task, primarily due to the sequence changes induced during the bisulfite conversion. For this purpose, we developed methGraph, a web application for easy, fast and flexible visualization and accurate in silico quality evaluation of PCR-based methylation assays. The visualization process starts by submitting PCR primer sequences for specificity assessment and mapping on the genome using the BiSearch ePCR primer-search algorithm. The next step comprises the selection of relevant UCSC genome annotation tracks for display in the final graph. A custom track showing all individual CpG dinucleotides, representing their distribution in the CpG island is also provided. Finally, methGraph creates a BED file that is automatically uploaded to the UCSC genome browser, after which the resulting image files are extracted and made available for visualization and download. The generated high-quality figures can easily be customized and exported for use in publications or presentations. methGraph is available at http://mellfire.ugent.be/methgraph/.     

Region of interest methylation analysis: a comparison of MSP with MS-HRM and direct BSP

The aim of this study was to compare and contrast three DNA methylation methods of a specific region of interest (ROI): methylation-specific PCR (MSP), methylation-sensitive high resolution melting (MS-HRM) and direct bisulfite sequencing (BSP). The methylation of a CpG area in the promoter region of Estrogen receptor alpha (ESR1) was evaluated by these three methods with samples and standards of different methylation percentages. MSP data were neither reproducible nor sensitive, and the assay was not specific due to non-specific binding of primers. MS-HRM was highly reproducible and a step forward into categorizing the methylation status of the samples as percent ranges. Direct BSP was the most informative method regarding methylation percentage of each CpG site. Though not perfect, it was reproducible and sensitive. We recommend the use of either method depending on the research question and target amplicon, and provided that the designed primers and expected amplicons are within recommendations. If the research question targets a limited number of CpG sites and simple yes/no results are enough, MSP may be attempted. For short amplicons that are crowded with CpG sites and of single melting domain, MS-HRM may be the method of choice though it only indicates the overall methylation percentage of the entire amplicon. Although the assay is highly reproducible, being semi-quantitative makes it of lesser interest to study ROI methylation of samples with little methylation differences. Direct BSP is a step forward as it gives information about the methylation percentage at each CpG site.     

Primer design versus PCR bias in methylation independent PCR amplifications

Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten “CpG-free” primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the “CpG-free” primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1–0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.     

MethyScan：一种甲基化特异性PCR引物设计及评估工具

DNA甲基化是重要的表观遗传现象,对基因表达发挥重要调控功能.大量研究表明,基因DNA甲基化是重要的临床诊断生物标志物.在临床上,实施快速、准确的DNA甲基化状态检测是诊断应用的前提和关键.甲基化特异性PCR(methylation specific PCR,MSP)通过将两种引物与甲基化、非甲基化模板各自特异性结合和扩增,实现基因甲基化状态的区分,是切实可行、简单便捷的临床诊断实验技术.但是,不同于常规PCR,MSP主要存在如何强化引物-甲基化/非甲基化模板特异性结合、降低引物序列Tm值差异、去除假阳性扩增及提高敏感性等四大难点.尽管大多数MSP引物设计软件对上述难题都提出了各自解决办法,但在引物设计影响因素考虑、设计与评估并行处理及特异性扩增预测等方面仍然存在较大缺陷.为此,本研究通过对MethPrimer、MSPPrimer、MethBlast、BiSearch等现有MSP引物设计软件原理的深入探究,以及对Bowtie、SAMtools和BEDTools等工具的有效综合整合,基于图形库Matplotlib和第三方Python功能库BioPython与Primer3-py实现了具有系列优点的甲基化特异性PCR引物设计与评估可视化工具MethyScan.它具有引物设计、基因组索引、引物评估等三大完整功能模块,不仅可快速进行MSP引物设计,实现巢式(Nested)引物适配,还可基于4种基因组碱基转换模板分析引物结合信息,图形化展示非特异性扩增与目的片段差异,从而综合评估引物特异性-非特异性扩增.同时,对食管癌、结直肠癌等多种恶性肿瘤中6个潜在生物标志物TFPI-2、NDRG4、CDKN2A、CD44、CASP8和SDHD的甲基化引物设计对比结果表明,MethyScan不仅可获得更多CpG位点的检测引物,而且所获得MSP引物位置与其他软件结果相同或相近,且引物间Tm值差值更小.总之,作为首个图形化展示特异性-非特异性扩增差异MSP引物设计工具,MethyScan可有效提高甲基化引物设计准确性,为临床DNA甲基化检测项目开展、检测试验实施及诊断试剂盒研发提供有力支撑.MethyScan工具下载地址:https://github.com/bioinfo-ibms-pumc/MethyScan.     

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.     

Disclosing Bias in Bisulfite Assay: MethPrimers Underestimate High DNA Methylation

Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of “Methylation-Insensitive Primers” (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.     

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.     

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that can be used for rapid quantitation of methylation at individual CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated Cytosine to Uracil while leaving 5-methylcytosine unaltered. Strand-specific PCR is performed to generate a DNA template for quantitative methylation analysis using Ms-SNuPE. SNuPE is then performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being interrogated. Reaction products are electrophoresed on polyacrylamide gels for visualization and quantitation by phosphorimage analysis. The Ms-SNuPE technique is similar to other quantitative assays that use bisulfite treatment of genomic DNA to discriminate unmethylated from methylated Cytosines (i.e., COBRA, pyrosequencing). Ms-SNuPE can be used for high-throughput methylation analysis and rapid quantitation of Cytosine methylation suitable for a wide range of biological investigations, such as checking aberrant methylation changes during tumorigenesis, monitoring methylation changes induced by DNA methylation inhibitors or for measuring hemimethylation. Approximately two to four CpG sites can be interrogated in up to 40 samples by Ms-SNuPE in less than 5 h, after PCR amplification of the desired target sequence and preparation of PCR amplicons.     

Epigenetic regulation of microRNAs in cancer: an integrated review of literature

MicroRNAs (miRNAs) belong to the heterogeneous class of non-coding RNAs (ncRNAs) that regulate the translation and degradation of target mRNAs, and control approximately 30% of human genes. MiRNA genes might be silenced in human tumors (oncomiRs) by aberrant hypermethylation of CpG islands that encompass or lie adjacent to miRNA genes and/or by histone modifications. We performed literature search for research articles describing epigenetically regulated miRNAs in cancer and identified 45 studies that were published between 2006 and 7/2010. The data from those papers are fragmented and methodologically heterogeneous and our work represents first systematic review towards to integration of diverse sets of information. We reviewed the methods used for detection of miRNA epigenetic regulation, which comprise bisulfite genomic sequencing PCR (BSP), bisulfite pyrosequencing, methylation specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation sensitive single nucleotide primer extension (Ms-SNuPE), MassARRAY technique and some modifications of those methods. This integrative study revealed 122 miRNAs that were reported to be epigenetically regulated in 23 cancer types. Compared to protein coding genes, human oncomiRs showed an order of magnitude higher methylation frequency (11.6%; 122/1048 known miRNAs). Nearly half, (45%; 55/122) epigenetically regulated miRNAs were associated with different cancer types, but other 55% (67/122) miRNAs were present in only one cancer type and therefore representing cancer-specific biomarker potential. The data integration revealed miRNA epigenomic hot spots on the chromosomes 1q, 7q, 11q, 14q and 19q. CpG island analysis of corresponding miRNA precursors (pre-miRNAs) revealed that 20% (26/133) of epigenetically regulated miRNAs had a CpG island within the range of 5kb upstream, among them 14% (19/133) of miRNAs resided within the CpG island. Our integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.     

Bisulfite-modified target DNA array for aberrant methylation analysis

Aberrant DNA methylation of CpG islands is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple and high-throughput methods of methylation analysis for earlier cancer diagnosis or the detection of recurrence. In this study, bisulfite-modified target DNA arrays were prepared on positively charged nylon membrane with two different procedures: fixing PCR products and fixing genomic DNA. First, a bisulfite PCR product array was prepared through fixing PCR products amplified in bisulfite sequencing primers from the bisulfite-modified genomic DNA of different clinical samples on membrane. Furthermore, bisulfite-modified genomic DNA of the different samples was directly fixed on membrane to fabricate bisulfite genomic DNA arrays. The two kinds of arrays were hybridized by probes labeled with digoxigenin, and the hybridization signals were obtained through chemiluminescent detection. The methylation statuses of the IGFBP7 gene for breast tumor and normal tissue samples and for normal human blood cell samples were detected successfully by the two procedures. It was shown that the methods are reliable and sensitive and that they have high potential in screening molecular methylation markers from a large number of clinical samples.     

Microarray-based Ms-SNuPE: near-quantitative analysis for a high-throughput DNA methylation

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In the present study, a microarray-based methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for parallel detecting changes of DNA methylation in cancer was developed. After modification by sodium sulfite, the unmethylated cytosine in the genomic DNA is converted to uracil while leaving the 5-methylcytosine unchanged, which can be detected by bifunctional primer carrying a unique sequence tag in addition to a locus-specific sequence. Because each locus has a distinct tag, the detecting reactions can be performed in a highly multiplexed fashion and the resulting product then be hybridized to the reverse complements of the sequence tags arrayed on a glass slide for methylation analysis. The calibration curves with the correlation coefficient >0.97 were established, which suggested that the method could be used in near-quantitative DNA methylation analysis. Two breast tumor-related genes (E-cad and p16) are successfully analyzed by two group primers (22 primers total), and the results are compatible with that of methylation-specific PCR (MSP). Our research proved that the method is simple and inexpensive, and could be applied as a high-throughput tool to quantitatively determine methylation status of the investigated genes.     

Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation

Methylation-specific PCR (MSP) is frequently used to distinguish methylated alleles in the genome. Sequences that have been incompletely converted during bisulfite treatment are frequently co-amplified during MSP. For accurate MSP, it is important to detect methylated sequences in a background of unconverted DNA with a high level of sensitivity. We report here sensitive techniques, bisulfite conversion-specific MSP (BS-MSP) to accurately evaluate CpG methylation. BS-MSP provides accurate results across a wide spectrum of bisulfite conversion levels. BS-MSP is also confirmed to be a useful technique for the routine analysis of clinical tumor specimens that were paraffin-embedded and microdissected. BS-MSP thus provides the powerful features of ease of use and compatibility with paraffin sections. We recommend that methylation analysis should include a step to eliminate unconverted DNA to avoid overestimation of the DNA methylation level in the samples.     

CpG PatternFinder: a Windows-based utility program for easy and rapid identification of the CpG methylation status of DNA

The bisulfite genomic sequencing technique is one of the most widely used techniques to study sequence-specific DNA methylation because of its unambiguous ability to reveal DNA methylation status to the order of a single nucleotide. One characteristic feature of the bisulfite genomic sequencing technique is that a number of sample sequence files will be produced from a single DNA sample. The PCR products of bisulfite-treated DNA samples cannot be sequenced directly because they are heterogeneous in nature; therefore they should be cloned into suitable plasmids and then sequenced. This procedure generates an enormous number of sample DNA sequence files as well as adding extra bases belonging to the plasmids to the sequence, which will cause problems in the final sequence comparison. Finding the methylation status for each CpG in each sample sequence is not an easy job. As a result CpG PatternFinder was developed for this purpose. The main functions of the CpG PatternFinder are: (i) to analyze the reference sequence to obtain CpG and non-CpG-C residue position information. (ii) To tailor sample sequence files (delete insertions and mark deletions from the sample sequence files) based on a configuration of ClustalW multiple alignment. (iii) To align sample sequence files with a reference file to obtain bisulfite conversion efficiency and CpG methylation status. And, (iv) to produce graphics, highlighted aligned sequence text and a summary report which can be easily exported to Microsoft Office suite. CpG PatternFinder is designed to operate cooperatively with BioEdit, a freeware on the internet. It can handle up to 100 files of sample DNA sequences simultaneously, and the total CpG pattern analysis process can be finished in minutes. CpG PatternFinder is an ideal software tool for DNA methylation studies to determine the differential methylation pattern in a large number of individuals in a population. Previously we developed the CpG Analyzer program; CpG PatternFinder is our further effort to create software tools for DNA methylation studies.     

Bisprimer--a program for the design of primers for bisulfite-based genomic sequencing of both plant and Mammalian DNA samples

Plants and animals differ in the sequence context of the methylated sites in DNA. Plants exhibit cytosine methylation in CG, CHG, and CHH sites, whereas CG methylation is the only form present in mammals (with an exception of the early embryonic development). This fact must be taken into account in the design of primers for bisulfite-based genomic sequencing because CHG and CHH sites can remain unmodified. Surprisingly, no user-friendly primer design program is publicly available that could be used to design primers in plants and to simultaneously check the properties of primers such as the potential for primer-dimer formation. For studies concentrating on particular DNA loci, the correct design of primers is crucial. The program, called BisPrimer, includes 2 different subprograms for the primer design, the first one for mammals and the second one for angiosperm plants. Each subprogram is divided into 2 variants. The first variant serves to design primers that preferentially bind to the bisulfite-modified primer-binding sites (C to U conversion). This type of primer preferentially amplifies the bisulfite-converted DNA strands. This feature can help to avoid problems connected with an incomplete bisulfite modification that can sometimes occur for technical reasons. The second variant is intended for the analysis of samples that are supposed to consist of a mixture of DNA molecules that have different levels of cytosine methylation (e.g., pollen DNA). In this case, the aim is to minimize the selection in favor of either less methylated or more methylated molecules.     

Methylation assay by nucleotide incorporation: a quantitative assay for regional CpG methylation density

Although the aberrant methylation in CpG islands is of great interest as a causative role in human malignancies, it has been very difficult to accurately determine methylation density. Here we report a novel microplate-based quantitative methylation assay, designated MANIC, for a region containing a number of CpG sites based on incorporation of hapten-labeled dCTP at cytosine sites where the methylated cytosines have not been converted to uracil by the bisulfite treatment. Validation using control DNAs revealed that the method was sensitive enough to detect < 1.25% methylated DNA and that calibration curve was linear. With this approach, we determined relative methylation density of O6-methylguanine-DNA methyltransferase gene promoter containing 12 CpG sites among the 12 colorectal cancers and corresponding normal mucosal tissues. Consequently, MANIC showed a high concordance with results by a quantitative method, bisulfite PCR single-stranded conformational polymorphism (BiPS). MANIC is a technique that avoids cumbersome procedures such as electrophoresis or the use of radiolabeling and is applicable to any sequence regardless of the total number of CpG sites or heterogeneity in methylation status.     

小鼠骨髓细胞中p16和Ralb基因启动区CpG岛的甲基化位点分析

抑癌基因p16和白血病致癌因子Ralb与白血病的发生密切相关,其启动子区CpG岛的甲基化对基因表达具有重要作用.本文旨在分析p16、Ralb基因启动子区CpG岛甲基化位点信息,并比较这两个基因在小鼠骨髓细胞和原代培养的骨髓细胞中甲基化状态的差异.运用"MethPrimer"软件预测p16、Ralb基因启动子区的CpG岛,设计甲基化特异性引物.利用重亚硫酸盐测序法(BSP)检测甲基化位点信息.结果显示,p16有1个CpG岛,岛上21个CpG位点全部未发生甲基化;Ralb有2个CpG岛,CpG岛1上的5个CpG位点全部呈甲基化状态,而CpG岛2上的17个CpG位点全部呈非甲基化状态,且小鼠骨髓细胞和体外原代培养的骨髓细胞中两基因的甲基化状态一致.表明p16、Ralb基因甲基化状态未受外界培养条件的影响而改变,提示在与两基因甲基化相关的研究中体外试验可替代体内试验.     

利用甲基化特异性引物高通量检测DNA甲基化

建立一种基于甲基化特异性引物和SAGE技术的高通量DNA甲基化定量检测新方法（MSP-SAGE）,首先利用亚硫酸氢钠对基因组DNA进行处理,使未甲基化的C转变为U,而甲基化的CpG不变.将处理和未处理的DNA双链变性后用随机引物PNNNNCG对存在含有CG的单链进行延伸,而无甲基化CG的单链处则不能延伸;将差异延伸的单链序列和频次信息经过系列分子操作后,引入PCR扩增模板;对中间带有未知序列的PCR扩增产物进行串连克隆测序.将来自于未处理组和处理组的某一CpG位点的序列出现的次数定义为［Tags］A和［Tags］B,将标准系列的实际甲基化水平和［Tags］B/［Tags］A之间建立线性回归方程.根据每一CpG位点的［Tags］B/［Tags］A比值可反推该位点的甲基化水平.MSP-SAGE具有良好的线性,基于标准系列的［Tags］B/［Tags］A与其实际甲基化水平的标准曲线方程为y＝1.455x（R²＝0.984,P<0.01）.MSP-SAGE的回收率在95%到110%之间,精确度位于4.2％和10.5%,检测限在3％左右,单次检测通量可达24个CpG位点.MSP-SAGE是一种很有应用前途的高通量DNA甲基化定量检测方法.     

A rapid, quantitative, non-radioactive bisulfite-SNuPE- IP RP HPLC assay for methylation analysis at specific CpG sites

The precise mapping and quantification of DNA methylation as an epigenetic parameter during development and in diseased tissues is of great importance for functional genomics. Here we describe a rapid, quantitative method to assess methylation levels at specific CpG sites using PCR products of bisulfite-treated genomic DNA. Using single nucleotide primer extension (SNuPE) assays in combination with ion pair reverse phase high performance liquid chromatography (IP RP HPLC) separation techniques, methylated and unmethylated CpGs can be discriminated and quantified based on the different masses and hydrophobicities of the extended primer products. The assay is linear, highly reproducible and several sites can be measured simultaneously in one reaction. It can be semi-automated and eliminates the need for cloning and sequencing of individual bisulfite PCR products.     

应用亚硫酸氢盐修饰后PCR联合TA克隆测序对E-cadherin启动子区甲基化水平的检测

E-cadherin是一种细胞粘附因子,通过增强细胞之间的粘附而起到抑制肿瘤转移的作用.Ecadherin基因启动子区的高甲基化是导致其在众多肿瘤细胞中表达下调甚至缺失的主要原因之一.本实验首先抽提SGC-7901细胞(胃腺癌细胞)、A549细胞(肺腺癌细胞)、MCF-7细胞(乳腺癌细胞)等3个肿瘤细胞株的全基因组DNA,然后对抽提的DNA进行亚硫酸氢盐修饰和纯化回收,根据修饰后的DNA序列设计引物并对其进行PCR扩增.然后将PCR扩增产物与pUC-T TA载体连接并转化入感受态大肠杆菌DH5α中进行培养,对筛选出的含有阳性重组子的菌落进行测序.测序结果显示,3个肿瘤细胞株的E-cadherin基因启动子区的CpG岛都呈现了高度的甲基化,亚硫酸氢盐的修饰效率达到了99.2%.综上研究表明,亚硫酸氢盐修饰后PCR(BSP)联合TA克隆测序可以对肿瘤细胞某基因启动子区CpG岛的甲基化水平进行精确量化,研究所使用的3个肿瘤细胞株均可作为研究肿瘤细胞E-cadherin基因甲基化的细胞模型.