Limitations and advantages of MS-HRM and bisulfite sequencing for single locus methylation studies.

The methylation-sensitive high-resolution melting (MS-HRM) protocol, as described by Wojdacz and Dobrovic, enables detection of a methylated template in an unmethylated background, with sensitivity similar to that of methylation-specific PCR (MSP). Furthermore, MS-HRM-based methylation screening is cost, labor and time efficient in contrast to direct bisulfite sequencing, which, therefore, is unsuitable as a screening method, but is still required to reveal the methylation status of individual CpG sites. In some experiments, detailed information on the methylation status of individual CpGs may be of interest for at least a subset of samples from MS-HRM-based methylation screening. For those samples, sequencing-based methodology has to be coupled with the MS-HRM protocol to investigate the methylation status of single CpG sites within the locus of interest. In this article, we review the limitations and advantages of MS-HRM and bisulfite sequencing protocols for single-locus methylation studies. Furthermore, we provide the insights into interpretation of the results obtained when a combination of the protocols is used for single-locus methylation studies.