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Abstract
Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.
Keywords: :
Acknowledgements
AD received grant support from the CLL Global Research Foundation, Victorian Cancer Agency, the National Breast Cancer Foundation of Australia, and the Cancer Council of Victoria. ILMC is supported by the Fay Marles scholarship from The University of Melbourne. The authors thank Giada Zapparoli for critical reading of the manuscript.
Figures and Tables
Figure 1 The effect on melting behavior of 5′-end biotin labeling of PCR primers. DNA methylation standards (100, 50, 25, 5 and 0% (WGA) methylation) were run in duplicate using primers (1) without biotin labeling (black curves), (2) with a biotin labeled forward primer (red curves) and (3) with a biotin labeled reverse primer (green curves). There does not appear to be any significant difference in melting behavior introduced by either biotin label. Replicates are grouped into a single line for clarity.
Figure 2 MS-HRM of the DAPK1 promoter for 10 CLL samples. (A) Normalized HRM curves. The DNA methylation standards of 0 (WGA), 25, 50 and 100% methylation are indicated. All amplicons from the CLL samples begin melting before the unmethylated control as can be seen by the earlier drop in fluorescence. Only samples 2 and 8 continue to melt after the unmethylated control has finished melting. (B) Tm plot (negative first derivative of the HRM curves). The broader peaks in the CLL samples that begin before the unmethylated control correspond to the earlier melting seen in (A), and result from heteroduplex formation. Only samples 2 and 8 encroach into the area under the peaks corresponding to methylation indicating the presence of more highly methylated templates.
Figure 3 Bisulfite pyrosequencing data. The MS-HRM products of the ten CLL samples were analyzed by bisulfite pyrosequencing. Pyrosequencing was performed on both the forward and reverse strands, following MS-HRM using one or the other of the biotin-labeled primers. The methylation percentage measured for each CpG dinucleotide obtained in each sequencing direction, the calculated means for each CpG dinucleotide of both sequencing reactions and the overall average calculated from all nine CpG positions of a given sample are shown. The individual methylation ratios as measured in both sequencing directions for each CpG position are similar. The heterogeneous methylation at an overall population level is revealed, with the average methylation level at each CpG dinucleotide given.
Figure 4 Representative pyrograms. DNA methylation level measured by bisulfite pyrosequencing of sample one in the forward (A) and reverse direction (B), respectively. The peaks used to determine the methylation ratio at each CpG dinucleotide are highlighted by light blue shading. The calculated methylation percentage for each CpG position is provided above the highlighted area. Nucleotides 44 and 7 are highlighted by light yellow shading in (A and B), respectively, and indicate control peaks to estimate incomplete bisulfite conversion. In (A), the CpG dinucleotides are analyzed from CpG 1 to CpG 9, whereas in (B), the CpG dinucleotides are analyzed from CpG 9 to CpG 1 because of the reverse sequencing of the upper strand of the PCR product.
Figure 5 Bisulfite sequencing of digitally obtained “clonal” PCR products. Five CLL samples representing the range of diversity of DNA methylation as determined by both the MS-HRM and the bisulfite pyrosequencing assays underwent bisulfite sequencing of digitally obtained “clonal” PCR products. Each horizontal line belongs to an epiallele. The CpG dinucleotides are represented by circles, where open and filled circles correspond to unmethylated and methylated CpG positions, respectively. The epiallelic resolution allows the determination of the DNA methylation patterns of each sample analyzed. The dMS-HRM amplicons encompass 16 CpG dinucleotides. The last nine CpG dinucleotides correspond to CpG positions 1 to 9 which were analyzed by the combined MS-HRM and bisulfite pyrosequencing approach. For clarity, sequencing data for just the last nine CpG dinucleotides is shown in this figure.
Figure 6 Comparison of bisulfite pyrosequencing with digital sequencing data. The mean DNA methylation values obtained for each CpG dinucleotide analyzed either by bisulfite pyrosequencing (from ) or digital Sanger sequencing (calculated from the individual epialleles of a given sample from ) are comparable, validating the combined MS-HRM and bisulfite pyrosequencing approach.
