GSE98921
Simultaneous methylation-level assessment of hundreds of CpG sites by targeted bisulfite PCR sequencing (TBPseq)
Methylated-DNA sequencing technologies are producing vast amounts of methylome data from cancer samples, from which cancer-associated differentially methylated CpG sites (cDMCs) are continuously identified and filed. The inclusion of as many cDMCs as possible helps improve the accuracy of cancer diagnosis and sometimes identify cancer subtypes. However, the lack of an established method for the analysis of hundreds of cDMCs practically impedes their robust use in clinical medicine. Here, we tested the availability of targeted bisulfite-PCR-sequencing (TBPseq) technology for the assessment of methylation levels of a myriad of CpGs scattered over the genome. In randomly selected 46 cancer cell lines, multiplexed PCR yielded a variety of amplicons harboring 250 CpGs residing at promoters of 97 cancer-associated genes, all of which were sequenced in the same flow cell. Clustering analysis of the TBPseq-assessed methylation levels of target CpGs showed that the lung and liver cancer cell lines correlated relatively strongly with each other while they weakly correlated with colon cancer cells. CpGs at the MLH1 gene promoter, which are known to be hypermethylated in colon cancers, indeed were heavily methylated in the tested colon cancer cells. LIFR CpGs, also known to be methylated in colon cancers, were hypermethylated in the colon cancer cells. Moreover, MLH1 and LIFR promoter hypermethylations were found in colon cancer cells only, but not in biliary tract, liver, lung and stomach cancers cell lines. A meta-analysis with public cancer methylome datasets verified the colorectal cancer specificity of MLH1 and LIFR promoter methylation. These results demonstrate that our TBPseq-based methylation assessment could be considered an effective, accurate, and competitive method to simultaneously examine a large number of target cDMCs and patient samples. Overall design: In randomly selected 46 cancer cell lines, multiplexed PCR yielded a variety of amplicons harboring 250 CpGs residing at promoters of 97 cancer-associated genes.