MBD-Seq as a Cost-Effective Approach for Methylome-Wide Association Studies: Demonstration in 1500 Case–Control Samples

Abstract

Aim We studied the use of methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) as a cost-effective screening tool for methylome-wide association studies (MWAS). Materials & methods Because MBD-seq has not yet been applied on a large scale, we first developed and tested a pipeline for data processing using 1500 schizophrenia cases and controls plus 75 technical replicates with an average of 68 million reads per sample. This involved the use of technical replicates to optimize quality control for multi- and duplicate-reads, an in silico experiment to identify CpGs in loci with alignment problems, CpG coverage calculations based on multiparametric estimates of the fragment size distribution, a two-stage adaptive algorithm to combine data from correlated adjacent CpG sites, principal component analyses to control for confounders and new software tailored to handle the large data set. Results We replicated MWAS findings in independent samples using a different technology that provided single base resolution. In an MWAS of age-related methylation changes, one of our top findings was a previously reported robust association involving GRIA2. Our results also suggested that owing to the many confounding effects, a considerable challenge in MWAS is to identify those effects that are informative about disease processes. Conclusion This study showed the potential of MBD-seq as a cost-effective tool in large-scale disease studies.

A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects

“…Our MWAS sequencing pipeline (35) and computational analysis methods (36,37) have been described previously, and the work flow for this project is summarized in Figure 1. After quality control (QC), our final study sample consisted of 718 subjects with, on average, 31.6 million reads per subject (SD ¼ 13.4 million).…”

“…We used the MethylMiner kit from Invitrogen, which employs MBD protein-based enrichment, to capture fragments with one or more methylated CpGs. Dependent on sample availability, we used 2-5 mg (mean ¼ 4.19 mg, SD ¼ 0.99 mg) of DNA starting material and eluted the captured fragments in 500 mM NaCl to increase the relative number of fragments from CpG poor regions (35), which otherwise would not be well covered (64). Methylated fragments were prepared for multiplexed single end (50 bp) sequencing on the SOLiD (Life Technologies) following the manufacturer's standard protocols.…”

“…To safeguard against errors arising from regions that are problematic to align, we previously performed an in silico alignment experiment, by fragmenting the reference genome and aligning it back to itself using standard alignment software (35). This experiment showed that 36% (10.5 million) of all genome-wide CpGs were in regions difficult to align, with the majority (71.8%) located in regions flagged as repetitive elements by RepeatMasker.…”

Crime investigation through DNA methylation analysis: methods and applications in forensics

“…Methyl-CpG binding domain protein sequencing (MBDseq) is another technique which is used to determine genome-wide 5mC methylation patterns of humans (Aberg et al, 2012). In MBD-seq method, genomic DNA is fragmented and the methylated sequences are pulled down by a 5mC binding protein or simply using MethylMiner Methylated DNA Enrichment Kit (Invitrogen) (Harris et al, 2010).…”

Clinical applicability of methylome sequencing in a pilot study of ischemic stroke individuals

“…While the number of clinical samples analysed is small, the findings of this evaluation suggest that MBD-seq is a suitable and sufficiently sensitive technology to determine methylation variability. The hypertension and plasma Hcy levels with genome-wide 5mC in a small group of ischemic stroke patients using methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) [9]. To the best of our knowledge, this is the first description of genome-wide MBD-seq to determine genomic methylation changes associated with cardiovascular disease risk.…”