DNA methylation profiling using the methylated-CpG island recovery assay (MIRA)

Abstract

The methylated-CpG island recovery assay (MIRA) exploits the intrinsic specificity and the high affinity of a methylated-CpG-binding protein complex (MBD2B and MBD3L1) to methylated CpG dinucleotides in genomic DNA. The MIRA approach works on double-stranded DNA and does not depend on the application of methylation-sensitive restriction enzymes. It can be performed on a few hundred nanograms of genomic DNA. Recently, the MIRA technique has been used to profile DNA methylation patterns at a resolution of 100 base pairs along the entire genome of normal human B-lymphocytes. The MIRA method is compatible with microarray and next generation DNA sequencing approaches. We describe the principles and details of this method applied for methylation profiling of genomes containing methylated CpG sequences.

Figure 1. MIRA-assisted genomic DNA methylation analysis

A schematic diagram of the basic MIRA approach is shown. For DNA fragmentation, we generally use sonication. Methylated DNA molecules are enriched in the MIRA procedure by binding to the MBD2B/MBD3L1 complex. In the microarray approach, input and MIRA-enriched (methylated) fractions are linker-ligated, amplified and labeled with different dyes, mixed, and hybridized to the slides, and the relative enrichment factors between different cell types are determined. Alternatively, MIRA-enriched DNA from two cell types can be compared directly. Open and closed circles represent methylated and unmethylated CpG dinucleotides, respectively; CGI, CpG island. In the high throughput sequencing approach (see Figure 2), the MIRA-enriched fractions are amplified in the Illumina cluster station after ligation of adaptors, and the ends are sequenced. Sequence reads are aligned to the genome.

Figure 2. MIRA sequencing

A. In the MIRA-seq approach, the methylated fragments of the genome are enriched by the MIRA technique and are sequenced using the Illumina Genome Analyzer. DNA obtained from a stage I lung squamous cell carcinoma was sonicated and the methylated fragments were collected by MIRA. Illumina adaptors were ligated, and the DNA analyzed on the Illumina Genome Analyzer. Using five lanes, ~15 million sequence reads were created and ~8 million uniquely mapped DNA sequence reads were aligned to the human genome. 1,254 of ~28,000 CpG islands had 30 or more mapped reads suggesting that they are methylated. The Figure shows an example of the sequence tags for the UNC5D gene aligned to the UCSC Genome Browser. The blue and red bars represent sequence reads of the forward and reverse strand, respectively. The peak maps to the CpG island (black box) near the 5′ end of the UNC5D gene. B. A NimbleGen tiling array for chromosome 8 was used to analyze the DNA of the same lung tumor. The peak maps to the CpG island (black box) near the 5′ end of the UNC5D gene.