Optimized design and data analysis of tag-based cytosine methylation assays

Suzuki, Masako; Qiang, Jun; Lia, Daniel; Pascual, Marien; McLellan, Andrew; Greally, John M.

doi:10.1186/gb-2010-11-4-r36

Cited by 79 publications

(117 citation statements)

References 26 publications

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“…We developed a high-throughput sequencing-based DNA methylation analysis, which consists of an expanded (more comprehensive) version of the methyl-sensitive cut counting assay (MSCC) (15,16). Applied to mouse, our method identifies the methylation status of 6 million CpGs (one-third of all existing CpGs) and covers 58% of the CpG-rich subsequences.…”

Section: Microbiologymentioning

confidence: 99%

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Expanded methyl-sensitive cut counting reveals hypomethylation as an epigenetic state that highlights functional sequences of the genome

Colaneri

Staffa

Fargo³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Methyl-sensitive cut counting (MSCC) with the HpaII methylation-sensitive restriction enzyme is a cost-effective method to pinpoint unmethylated CpGs at single base-pair resolution. However, it has the drawback of addressing only CpGs in the context of the CCGG site, leaving out the remainder of the possible 16 XCGX tetranucleotides in which CpGs are found. We expanded MSCC to include three additional enzymes to address a total of 5 of the 16 XCGX combinations. This allowed us to survey methylation at about one-third of all a mammalian genome's CpGs. Applied to mouse liver DNA, we correctly confirmed data reported with other methods showing hypomethylation to be concentrated at promoters and in CpG islands (CGIs), with gene bodies and intergenic regions being mostly methylated. Grouping unmethylated CpGs, characterized by high MSCC scores (7% false discovery rate), we found a large number of unmethylated regions not qualifying as CGIs located in intergenic and intronic regions, which are highly enriched in functional DNA sequences (open regulatory annotation database) as well as in noncoding yet highly conserved mammalian sequences thought to be important but with as yet unknown function. About 50% of MSCC-defined unmethylated regions do not overlap algorithm-defined CGIs and offer a novel search space in which new functionalities of DNA may be found in health and disease.

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Section: Microbiologymentioning

confidence: 99%

“…On page 20642, left column, within the Abstract, lines [16][17][18], "Finally, we describe the extension of the dienamine catalysisinduced vinylogous nucleophilicity to the asymmetric γ-amination of cyclohexene carbaldehyde" should be removed from the article.…”

mentioning

confidence: 99%

Expanded methyl-sensitive cut counting reveals hypomethylation as an epigenetic state that highlights functional sequences of the genome

Colaneri

Staffa

Fargo³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…12 Genomic representations by the methylationsensitive HpaII enzyme are more accurately reflective of DNA methylation when compared with its methylationinsensitive isoschizomer MspI, which controls for differences in the ability of individual loci to be amplified. In tumors, gains and losses of chromosome regions can confound many genome-wide assays, but the concurrent generation of the MspI representation from our osteosarcoma samples allows adjustment to account for this source of variability.…”

Section: Resultsmentioning

confidence: 99%

“…To control for differing numbers of total reads for each sample, DNA methylation values expressed as angle values were calculated as previously described 12 after rescaling the log and z transformations of the raw HpaII and MspI counts. Data are available on the Gene Expression Omnibus (GSE59200).…”

Section: Discussionmentioning

confidence: 99%

“…12 Genomic DNA was digested with either the methylation sensitive restriction enzyme, HpaII, or its methylation insensitive isoschizomer, MspI. An Illumina sequencing adapter containing a T7 transcriptional promoter and an EcoP15I restriction site at the 3 0 end was ligated to the digested DNA, enabling digestion with EcoP15I and the generation of a 27 base pair genomic DNA fragment.…”

Section: Methodsmentioning

confidence: 99%

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Predictive properties of DNA methylation patterns in primary tumor samples for osteosarcoma relapse status

et al. 2015

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Osteosarcoma is the most common primary malignant bone tumor in children. Validated biological markers for disease prognosis available at diagnosis are lacking. No genome-wide DNA methylation studies linked to clinical outcomes have been reported in osteosarcoma to the best of our knowledge. To address this, we tested the methylome at over 1.1 million loci in 15 osteosarcoma biopsy samples obtained prior to the initiation of therapy and correlated these molecular data with disease outcomes. At more than 17% of the tested loci, samples obtained from patients who experienced disease relapse were more methylated than those from patients who did not have recurrence while patients who did not experience disease relapse had more DNA methylation at fewer than 1%. In samples from patients who went on to have recurrent disease, increased DNA methylation was found at gene bodies, intergenic regions and empirically-annotated candidate enhancers, whereas candidate gene promoters were unusual for a more balanced distribution of increased and decreased DNA methylation with 6.6% of gene promoter loci being more methylated and 2% of promoter loci being less methylated in patients with disease relapse. A locus at the TLR4 gene demonstrates one of strongest associations between DNA methylation and 5 y event-free survival (P-value D 1.7 £ 10 ¡6 ), with empirical annotation of this locus showing promoter characteristics. Our data indicate that DNA methylation information has the potential to be predictive of outcome in pediatric osteosarcoma, and that both promoters and non-promoter loci are potentially informative in DNA methylation studies.

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