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

Abstract

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.

Keywords::

• biomarker
• cancer
• diagnostics
• method
• methylation
• methylation-sensitive high-resolution melting
• Sanger sequencing

Acknowledgements

The authors would like to acknowledge Tina Kjeldsen for the excellent technical assistance with the experiments. Henrik Hager kindly donated the clinical specimens used in the experiments. This work was supported by the Lundbeck, Toyota and Harboe Foundations. The authors also would like to thank Roche Diagnostics for the support of their research.

Financial & competing interests disclosure

None of the aspects of this publication has been discussed with Roche Diagnostics. Tomasz K Wojdacz and Lise Lotte Hansen are listed coinventors on the patent pending on the aspects of MS-HRM protocol. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.