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.     

Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling

Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.     

Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution

Background  

New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed.     

Heterogeneity and randomness of DNA methylation patterns in human embryonic stem cells

DNA methylation has been proposed to be important in many biological processes and is the subject of intense study. Traditional bisulfite genomic sequencing allows detailed high-resolution methylation pattern analysis of each molecule with haplotype information across a few hundred bases at each locus, but lacks the capacity to gather voluminous data. Although recent technological developments are aimed at assessing DNA methylation patterns in a high-throughput manner across the genome, the haplotype information cannot be accurately assembled when the sequencing reads are short or when each hybridization target only includes one or two cytosine-phosphate-guanine (CpG) sites. Whether a distinct and nonrandom DNA methylation pattern is present at a given locus is difficult to discern without the haplotype information, and the DNA methylation patterns are much less apparent because the data are often obtained only as methylation frequencies at each CpG site with some of these methods. It would facilitate the interpretation of data obtained from high-throughput bisulfite sequencing if the loci with nonrandom DNA methylation patterns could be distinguished from those that are randomly methylated. In this study, we carried out traditional genomic bisulfite sequencing using the normal diploid human embryonic stem (hES) cell lines, and utilized Hamming distance analysis to evaluate the existence of a distinct and nonrandom DNA methylation pattern at each locus studied. Our findings suggest that Hamming distance is a simple, quick, and useful tool to identify loci with nonrandom DNA methylation patterns and may be utilized to discern links between biological changes and DNA methylation patterns in the high-throughput bisulfite sequencing data sets.     

High density DNA methylation array with single CpG site resolution

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R² of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.     

癌症DNA甲基化调控位点的识别

DNA甲基化是一种重要的表观遗传学修饰,在基因的转录调控方面具有重要的作用。异常的DNA甲基化可以导致癌症等复杂疾病发生,癌基因相关的DNA甲基化调控位点的识别对于解析癌症的发生发展机制及识别新的癌症标记具有重要意义。本研究通过整合The Cancer Genome Atlas(TCGA)的泛癌症基因组的高通量甲基化谱和基因表达谱,识别癌基因相关的DNA甲基化调控位点。对于每种癌症分批次计算Cp G位点甲基化与相关基因表达之间的相关性,并筛选调控下游基因的Cp G位点(包括强调控位点、弱调控位点和不调控位点),结果表明仅有一半的Cp G位点对下游基因具有调控作用;对癌症间共享的调控位点的分析发现不同癌症间共享的调控位点不尽相同,表明癌症特异的甲基化调控位点的存在。进一步地,对差异甲基化和差异表达基因的功能富集分析揭示了受甲基化调控的基因确实参与了癌症发生发展相关的功能。本研究的结果是对当前甲基化调控位点集的重要补充,也是识别癌症新型分子标记特征的重要资源。     

Methyl-DNA immunoprecipitation (MeDIP): hunting down the DNA methylome

One of the most challenging projects in the field of epigenetics is the generation of detailed functional maps of DNA methylation in different cell and tissue types in normal and disease-associated conditions. This information will help us not only understand the role of DNA methylation but also identify targets for therapeutic treatment. The completion of the various epigenome projects depends on the design of novel strategies to survey and generate detailed cartograms of the DNA methylome. Methyl-DNA immunoprecipitation (MeDIP) assays, in combination with hybridization on high-resolution microarrays or high-throughput sequencing (HTS) techniques, are excellent methods for identifying methylated CpG-rich sequences. We provide a critical overview of different genome-wide techniques for DNA methylation analysis and propose that MeDIP assays may constitute a key method for elucidating the hypermethylome of cancer cells.     

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.     

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.     

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.     

Quantitative methylation analysis using methylation-sensitive single-nucleotide primer extension (Ms-SNuPE)

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that allows for rapid and simultaneous quantitation of the degree of methylation at several CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. A strand-specific polymerase chain reaction product is then generated to provide a suitable DNA template for quantitative methylation analysis using Ms-SNuPE. Single-nucleotide primer extension is performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being analyzed. The Ms-SNuPE technique can be adapted for high-throughput methylation analysis and therefore represents a novel approach for rapid quantitation of cytosine methylation suitable for a wide range of biological investigations.     

Restriction landmark genome scanning

Restriction landmark genome scanning (RLGS) is a quantitative approach that is uniquely suited for simultaneously assessing the methylation status of thousands of CpG islands. RLGS separates radiolabeled NotI fragments (or other CpG-containing restriction enzyme fragments) in two dimensions and allows distinction of single-copy CpG islands from multicopy CpG-rich sequences. The methylation sensitivity of the endonuclease activity of NotI provides the basis for differential methylation analysis, and NotI sites occur primarily in CpG islands and genes. RLGS has been used to identify novel imprinted genes, novel targets of DNA amplification and methylation in human cancer, and to identify deletion, methylation, and gene amplification in a mouse model of tumorigenesis. Such massively parallel analyses are critical for pattern recognition within and between tumor types, and for estimating the overall influence of CpG island methylation on the cancer cell genome. RLGS is also a useful method for integrating methylation analyses with high-resolution gene copy number analyses.     

Screening for and analysis of methylation differences using methylation-sensitive single-strand conformation analysis

Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a method of screening for methylation changes at CpG sites in a region of DNA. After bisulfite modification, the region of interest is amplified using primers specific for bisulfite-modified sequences. The amplified products are denatured and run on a nondenaturing polyacrylamide gel. The sequence differences caused by methylation lead to the formation of different secondary structures (conformers) with different mobilities. MS-SSCA is a convenient and rapid method for screening large numbers of samples for methylation. Individual bands can readily be isolated and sequenced allowing more detailed analysis of methylation changes. In this article, we present a protocol for MS-SSCA and outline strategies for the design of primers for amplifying bisulfite-modified DNA sequences.     

Detection method for methylation density on microarray using methyl-CpG-binding domain protein

A method for determining methylation density of target CpG islands has been established. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite-converted sample DNAs. The PCR products on the microarray were treated by SssI methyltransferase and labeled with TAMRA fluorescence. A recombinant, antibody-like methyl-CpG-binding protein labeled with Cy5 fluorescence was used to identify symmetrical methyl-CpG dinucleotide of the PCR products on the microarray. By use of a standard curve with control mixtures, the ratio of two fluorescence signals can be converted into percentage values to assess methylation density of targeted fragments. We obtained the methylation density of six CpG islands on the two tumor suppressor genes of CDK2A and CDK2B from seven cancer cell line samples and two normal blood samples. The validity of this method was tested by bisulfite sequencing. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples.     

Analysis of repetitive element DNA methylation by MethyLight

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.     

Assaying DNA methylation based on high-throughput melting curve approaches

Here we describe two high-throughput methods to assay DNA methylation, melting curve methylation specific PCR (McMSP) and melting curve combined bisulfite restriction analysis (McCOBRA), which adapt standard MSP and COBRA methods to a melting curve analysis based platform. We show that McMSP and McCOBRA can accurately determine methylation status in a high-throughput and gel-free manner. Moreover, McCOBRA can be used to quantitatively estimate the percent of methylated DNA at a specific CpG site within a heterogeneous sample. The accuracy of McMSP and McCOBRA was initially tested using the 5'-CpG site of the tumor-suppressor gene CDKN2A as a model system in homogeneous and heterogeneous controls, and cancer cell line samples. Furthermore, the robustness of McMSP and McCOBRA was validated in four additional loci. We demonstrate that McCOBRA and McMSP provide several advantages over existing methods, as they are simple, accurate, and high-throughput, which makes them widely applicable to large-scale methylation studies.     

High-throughput assay of DNA methylation based on methylation-specific primer and SAGE

Mapping of genomic DNA methylation is a dispensable part of functional genome. We have developed a novel method based on methylation-specific primer and serial analysis of gene expression, called MSP-SAGE, with potential of high-throughput quantification of genomic DNA methylation. We used a 6-mer methylation-specific primer to extend the methylated CpG sequences other than non-methylated CpG sequences. The 17 bp tags contained methylated CpG sequence, which were obtained from extended methylation sequence by digestion of restriction endonuclease, and then the tags were concatenated and cloned for sequencing. We can identify the locations of methylation according to the sequences of tags and quantify the methylation status from the frequency of the tags. MSP-SAGE has a good linearity in a broad methylation range from 5% to 100% with good accuracy and high precision. The proof-of-principle study shows that MSP-SAGE is a reliable high-throughput assay for quantification of DNA methylation.