Quality evaluation of methyl binding domain based kits for enrichment DNA-methylation sequencing

DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD) based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1) to identify a best performing kit for MBD-based enrichment using independent validation data, and 2) to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3). Reduced representation bisulfite sequencing data (all three cell lines) and publicly available Illumina Infinium BeadChip data (DU145 and PC3) were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode's MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications

Fragment CpG-plots.

<p>Fractions of mapped MBD-seq fragments with different CpG-counts for cell lines HCT15 (A), DU145 (B) and PC3 (C) for the different kits: MethylMagnet (violet), MethylCollector (yellow), MethylCollector Ultra (MC Ultra, blue), MethylCap (red) and MethylMiner (green).</p

Exploratory comparison of MBD-seq and RRBS data.

<p>Visual comparison of MBD-seq results for MethylMagnet (MMag), MethylCollector (MCol), MethylCollector Ultra (MCU), MethylCap (MCap) and MethylMiner (MMin) with RRBS data for the promoter regions of four selected loci, i.e. <i>Igfbp3</i> (panel A, chromosome 7, depicted from position 45959883 to 45962146), <i>Tert</i> (panel B, chromosome 5, 1293850 to 1296219), <i>Epb41l3</i> (panel C, chromosome 8, 5628365 to 5630973) and <i>Socs3</i> (panel D, chromosome 17, 76354158 to 76357420). CpGs assessed by RRBS are indicated as vertical red (fraction methylated)/grey (fraction unmethylated) bars. Note that RRBS only assesses the methylation status of a (sometimes variable) subfraction of CpGs.</p

Yield independent genome-wide kit evaluation using RRBS data for external validation.

<p>Fractions of mapped MBD-seq fragments corresponding with specific RRBS methylation degrees (binned per 2%) for the different cell lines (A, HCT15; B, DU145; C, PC3) and kits (violet, MethylMagnet; yellow, MethylCollector; blue, MethylCollector Ultra (MC Ultra); red, MethylCap; green, MethylMiner) with indication of the background profile (black, fractions of all RRBS values measured for specific cell line). Additionally, the same fractions after division by the corresponding background profile fractions are plotted (D, HCT15; E, DU145; F, PC3).</p

Yield independent genome-wide kit evaluation using Infinium HumanMethylation27 BeadChip data for external validation.

<p>Fractions of mapped MBD-seq fragments corresponding with specific Infinium methylation degrees (binned per 5%) for two cell lines (A, DU145; B, PC3) and all kits (violet, MethylMagnet; yellow, MethylCollector; blue, MethylCollector Ultra (MC Ultra); red, MethylCap; green, MethylMiner) with indication of the background profile (black, fractions of all Infinium methylation values measured for specific cell line). Additionally, the same fractions after division by the corresponding background profile fractions are plotted (C, DU145; D, PC3).</p