The authors examined single-base level DNA methylation in embryonic stem cells (H1) and fibroblasts (IMR90), utilizing MethylC-Seq/ChIP-Seq and the Genome Analyzer II. Nearly all methylcytosines in the IMR90 genome were CG; almost 25% of H1 had non-CG methylation. This was mostly lost with H1 cell differentiation. At the mCG sites IMR90 cells were less frequently methylated; methylation also differed by chromosomal location. Highly expressed genes had three times the non-CG methylation density than non-expressed genes.

DNA methylation at CpG ‘islands’ (CGIs) may induce problematic gene expression. Current profiling investigates single nucleotides within limited regions, or whole genomes with limited resolution. The bisulfite capture method combined bisulfite conversion, hybrid selection, and deep sequencing for methylation frequencies in 324 CGIs, using Genome Analyzer II. Chip-Seq was used to investigate histone modification. In a breast cancer versus normal skin cell line there was a higher frequency of full or partial methylation, and enriched methylated sites within exons.

Many diseases, including cancer, involve CpG mutations. Cost-effective sequencing can be achieved using multiplexed targeted amplification. Here padlock capturing technology efficiency, sensitivity, and uniformity was vastly improved. The authors designed padlock probes covering 24% of CpGs on chromosome 21. They sequenced libraries on the Genome Analyzer II. Efficiency in the capture of 55,000 exons was increased 10,000 fold by extending the genomic DNA/padlock probe hybridization reaction time, increasing amounts of padlock probes, and optimization of dNTP concentrations.

The authors investigated epigenetic DNA methylation in the endosperm and embryo of Arabidopsis during development, with the aid of the Genome Analyzer. They had a number of findings, including that the highest levels of methylation were closer to centromeres, that genes encoding proteins were generally less methylated than those encoding transposable elements, and that methylation was lower in the endosperm compared to the embryo. The also found five previously unknown imprinted genes preferably expressed in the endosperm.

Using high-throughput bisulfite sequencing on the Genome Analyzer, the authors show widespread DNA demethylation in Arabidopsis thaliana endosperm. This was found to be at least partially controlled by the maternal DNA glycosylase DEMETER as mutations of this somewhat restored CG methylation in this area.

Epigenetic DNA methylation predominantly occurs at cytosine residues in the CpG dinucleotide. Sodium bisulfite-related analysis relies on it’s preserving of methylated cytosines Investigation of genome-wide DNA methylation in the mouse (Mus musculus) was carried out through optimization of reduced representation bisulfite sequencing (RRBS), which generates nucleotide resolution Illumina-based libraries using the Genome Analyzer. RRBS is similar to BS-seq but it used CpG-specific endonucleases to digest genomic DNA and provides enhanced coverage.

The authors examined cytosine methylation using massively parallel sequencing on the Genome Analyzer. They developed bisulfite padlock (molecular inversion) probes (BSPP) to target CpG locations (not necessarily concentrated in CpG islands or promoter regions) in human B-lymphocytes, fibroblasts and induced pluripotent stem cells. They also used methyl-sensitive cut counting (MSCC) to generate genome-wide, nontargeted, data in the DNA of B-lymphocytes. In highly expressed genes there was high methylation in the gene-body with low methylation in the promoter region.

Investigation of single-nucleotide DNA methylation was carried out on arbitrary targets on chromosomes 12, 20, and 34. They used ~30,000 bisulfite padlock probes (BSPP) to study 2,020 CpG islands in human fibroblast and pluripotent stem cell lines and found slightly enhanced chromosome-wide cytosine methylation in the latter. Used Genome Analyzer for single-molecule bisulfite sequencing.

The authors developed a DNA-methylation assay (Methyl-seq) to examine over 90,000 regions using human embryonic stem cells (hESCs), their derivatives, and liver samples. In Methyl-seq, DNA is digested with methyl-sensitive restriction enzymes to enable creation of a fragment library for sequencing by the Genome Analyzer. They found differentiation resulted in 2-11% DNA methylation changes. hESC differentiation has both methylation (at transcription-related sites) and demethylation, with only the latter, at transcription-related sites, in fetal liver development.