Cell-free DNA (
cfDNA) from patient blood is emerging as a noninvasive diagnostic avenue for various
cancers. We aimed to identify reliable
biomarkers in
cfDNA by investigating genes exhibiting significant differences between
colorectal cancer and control samples. Our objective was to identify genes that showed a positive difference between
cancer and control samples. To achieve this, we conducted an in silico analysis to identify genes that exhibit no significant variation in methylation between genomic
DNA (gDNA) and
cfDNA. We collected experimental data from publicly available repositories, which included
5-hydroxymethylcytosine (5hmC) profiles of gDNA and
cfDNA samples from both
cancer patients and healthy individuals. By comparing and overlapping these two groups, we identified 187 genes of interest, of which 53 genes had a positive difference among
colon cancer patients and healthy individuals. Next, we performed an ANOVA test on these genes, resulting in the identification of 12 genes that showed statistically significant higher levels of 5hmC in
cfDNA and gDNA from
cancer patients compared to healthy individuals. Additionally, we compared the 5hmC status of these genes between
cfDNA and gDNA from
cancer patients. Interestingly, we found that the 5hmC of the
toll like receptor 4 (TLR4) gene was not statistically different between
cfDNA and gDNA from
cancer patients, indicating consistency between
cfDNA and gDNA. These findings have important implications, not only for experimental validation but also for the development of more sensitive and robust noninvasive methods to improve diagnostic, prognostic, and treatment options for
colon cancer.