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BSP/MSP
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Highly sensitive.
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Gives rise to false positivity if bisulfite modification is incomplete.
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Highly specific for particular CpG sites.
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Poor design of primers can give rise to inconclusive results.
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Facilitates the analysis of clinical samples with low levels of methylated sequences.
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Low multiplexing capabilities.
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Obviates the use of restriction enzymes and eliminates the problem of incomplete enzyme digestion.
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Need for methylation specific probes in addition to primers—when conducting BSP and hybridization probe-based distinction of ME/UM sequences.
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Bisulfite sequencing
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Highly specific. Usually depending on BSP or MSP before sequencing.
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Technically demanding; similar to MSP/BSP because depending on BSP amplification.
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Delineates the methylation status of each individual CpG site.
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Time consuming and labor intensive.
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Requires very low DNA amounts.
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Gives rise to false positivity if bisulfite modification is incomplete.
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NGS-based high multiplexing capability when pooling single-MSP-derived amplicons.
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MSRE
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High sensitivity and enables high multiplexing and quantitative read out.
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Limited to target regions, which are covered by MSRE recognition sequence.
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Detection of low fraction (0.1-x%) of methylated in unmethylated DNA background.
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Gives rise to false positivity if enzyme digestion is incomplete.
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Suitable for multiplexed analyses 50–100 amplicons starting from ng-amounts (e.g., cfDNA).
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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.
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