Table 1 Comparison of different DNA methylation analysis methods

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.