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A box and whisker plot was generated in R to display the distribution of DNA methylation ratios between polyp and germline tissues across the DNMT3L promoter.
Next, we compared the tumor/normal methylation ratios between different groups of patients.
Roughly 10% of the CpGs were completely unmethylated (methylation ratio <0.05), while 40% of the CpG dinucleotides showed partial methylation ratios between 0.05 and 0.95.
Fisher's exact test was used to identify 3,004,806 CpG dinucleotides with a statistically significant (P <0.05) difference in their methylation ratios between the young and the old datasets.
Differentially methylated cytosines were discovered using a Fisher Exact Test, with a p-value threshold of 0.05, a minimum depth of 3, and a minimum of 33%% nominal difference in methylation ratios between conditions.
Comparing the methylation ratios between the matched self- and physician-taken samples showed again a very high concordance (P<0.001), with the best results for C13ORF18 (r=0.82, P<0.001), EPB41L3 (r=0.84, P<0.001) and JAM3 (r=0.89, P<0.001) methylation.
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(C ) Metagene plots of CpG methylation ratio between the mutant and its wt counterpart.
However, methylation ratio between T21 and normal placental samples was not different (P > 0.05 for all CpG sites).
However, in general, a clear correlation between methylation ratios in normal and cancerous tissues could be detected.
Likewise, the relationship between methylation ratios and standard clinicopathological variables (age, serum PSA levels, tumor grade and stage), were evaluated using the Kruskall-Wallis or Mann–Whitney tests.
Procedure reproducibility was shown by a strong positive correlation (r = 0.94) between the fragment coverage of both MEBS libraries as well as between the methylation ratios of all C positions covered in both libraries (r = 0.94).
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