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Table 1 Comparison of different DNA methylation analysis methods
| Method | Advantages | Limitations |
|---|---|---|
| BSP/MSP | Highly sensitive. | Gives rise to false positivity if bisulfite modification is incomplete. |
| Highly specific for particular CpG sites. | Poor design of primers can give rise to inconclusive results. | |
| Facilitates the analysis of clinical samples with low levels of methylated sequences. | Low multiplexing capabilities. | |
| Obviates the use of restriction enzymes and eliminates the problem of incomplete enzyme digestion. | Need for methylation specific probes in addition to primers—when conducting BSP and hybridization probe-based distinction of ME/UM sequences. | |
| Bisulfite sequencing | Highly specific. Usually depending on BSP or MSP before sequencing. | Technically demanding; similar to MSP/BSP because depending on BSP amplification. |
| Delineates the methylation status of each individual CpG site. | Time consuming and labor intensive. | |
| Requires very low DNA amounts. | Gives rise to false positivity if bisulfite modification is incomplete. | |
| NGS-based high multiplexing capability when pooling single-MSP-derived amplicons. | ||
| MSRE | High sensitivity and enables high multiplexing and quantitative read out. | Limited to target regions, which are covered by MSRE recognition sequence. |
| Detection of low fraction (0.1-x%) of methylated in unmethylated DNA background. | Gives rise to false positivity if enzyme digestion is incomplete. | |
| Suitable for multiplexed analyses 50–100 amplicons starting from ng-amounts (e.g., cfDNA). | Internal standards recommended because no direct distinction of ME/UM alleles is feasible based on the sequence (like C vs T upon BS-based analysis in sequences) when aiming NGS-based readout. |
