Abstract
BackgroundDNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer. The quantitative analysis of DNA methylation patterns has the potential to serve as diagnostic and prognostic biomarkers, however, there is currently a lack of consensus regarding the optimal methodologies to quantify methylation status. To address this issue we compared five analytical methods: (i) MethyLight qPCR, (ii) MethyLight digital PCR (dPCR), methylation-sensitive and -dependent restriction enzyme (MSRE/MDRE) digestion followed by (iii) qPCR or (iv) dPCR, and (v) bisulfite amplicon next generation sequencing (NGS). The techniques were evaluated for linearity, accuracy and precision.ResultsMethyLight qPCR displayed the best linearity across the range of tested samples. Observed methylation measured by MethyLight- and MSRE/MDRE-qPCR and -dPCR were not significantly different to expected values whilst bisulfite amplicon NGS analysis over-estimated methylation content. Bisulfite amplicon NGS showed good precision, whilst the lower precision of qPCR and dPCR analysis precluded discrimination of differences of < 25% in methylation status. A novel dPCR MethyLight assay is also described as a potential method for absolute quantification that simultaneously measures both sense and antisense DNA strands following bisulfite treatment.ConclusionsOur findings comprise a comprehensive benchmark for the quantitative accuracy of key methods for methylation analysis and demonstrate their applicability to the quantification of circulating tumour DNA biomarkers by using sample concentrations that are representative of typical clinical isolates.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1174) contains supplementary material, which is available to authorized users.
Highlights
DNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer
To assess the accuracy and precision of DNA methylation quantification technologies, five commonly used methods for the quantification of the proportion of methylated DNA molecules were investigated by analysing the methylation status of the biomarker, p14ARF, with each method targeting the same region of the p14ARF promoter (Additional file 1), using a panel of DNA standards combined in a range of methylated and unmethylated ratios
Conclusions previous investigations have discussed the relative merits of bisulfite conversion vs. Restriction enzyme (RE) digestion based methods of methylation quantification [18,24,25], this is the first study to directly compare both upstream treatments with alternative downstream PCR methods and platforms
Summary
DNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer. The quantitative analysis of DNA methylation patterns has the potential to serve as diagnostic and prognostic biomarkers, there is currently a lack of consensus regarding the optimal methodologies to quantify methylation status. To address this issue we compared five analytical methods: (i) MethyLight qPCR, (ii) MethyLight digital PCR (dPCR), methylation-sensitive and -dependent restriction enzyme (MSRE/MDRE) digestion followed by (iii) qPCR or (iv) dPCR, and (v) bisulfite amplicon generation sequencing (NGS). MethyLight [18,19] is one of the most common PCR based approaches; it involves the bisulfite conversion of DNA followed by real-time quantitative PCR (qPCR) by using primers and hydrolysis probes that are complementary to either the methylated or unmethylated bisulfite-converted DNA sequences. Recent studies have applied RE digestion analysis to dPCR platforms [26,27], with Hindson et al [26] demonstrating a superior precision and sensitivity of measurement compared to qPCR
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