Quality Evaluation of Methyl Binding Domain Based Kits for Enrichment DNA-Methylation Sequencing

Abstract

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.

Methyl-CpG/MBD2 Interaction Requires Minimum Separation and Exhibits Minimal Sequence Specificity

“…This result builds on previous work that has reported on read coverage of the MethylMiner kit as a function of local CpG density (24,25) and CpG number (16), noting low sensitivity to sparsely methylated DNA. Our data uniquely analyzes the explicit property of the DNA fragment (CpG number) with high certainty of methylation level (M.SssI treatment) while accounting for the background frequency of reads with a given CpG count (dividing the fraction of pulldown by the fraction of input) and resolving the bias for less frequent, highly methylated reads.…”

“…As the name suggests, this family of proteins contains a methyl-binding domain that allows it to preferentially bind to a methylated CpG (mCpG) in DNA. Of the known MBD proteins, MBD2 discriminates most strongly between mCpGs and other genetic sequences (14,15) and has been incorporated into several commercially available kits for methylation detection (16). A typical MBD pulldown experiment for enrichment of methylated DNA consists of: (1) fragmentation of genomic DNA, (2) introduction of the fragments to the magnetic microbeads coated in biotinylated MBD proteins, (3) an incubation period during which the MBD proteins interact with the DNA fragments and potentially bind, (4) pulldown of the beads (and the protein-bound fragments), (5) elution to free the fragments from the proteins, (6) sequencing of the fragments, and finally (7) an alignment of those fragments to a reference genome.…”

Morphine Induces Redox-Based Changes in Global DNA Methylation and Retrotransposon Transcription by Inhibition of Excitatory Amino Acid Transporter Type 3–Mediated Cysteine Uptake

“…Genomic DNA was extracted from samples with the Easy DNA kit (K1800-01; Invitrogen, Carlsbad, CA) using the appropriate protocol for cell lines. For a full overview of the MethylCap-Seq protocol, refer to De Meyer et al (2013). In summary, fragmentation was performed with a Covaris S2 (Woburn, MA) with the following settings: duty cycle 10%, intensity 5, 200 cycles per burst during 200 seconds, to obtain fragments with an average length of 200 bp.…”

SNP-guided identification of monoallelic DNA-methylation events from enrichment-based sequencing data

“…The samples were sequenced according to the protocol described in the paper of De Meyer et al (29) with some additional modifications: (i) After DNA fragmentation, the methylated fragments PAGE 3 OF 14 Nucleic Acids Research, 2014, Vol. 42, No.…”