title: nMETR: technique for facile recovery of hypomethylation genomic tags.
abstract: Genome-wide methylation studies frequently lack adequate controls to estimate proportions of background reads in the resulting datasets. To generate appropriate control pools, we developed technique termed nMETR (non-methylated tag recovery) based on digestion of genomic DNA with methylation-sensitive restriction enzyme, ligation of adapter oligonucleotide and PCR amplification of non-methylated sites associated with genomic repetitive elements. The protocol takes only two working days to generate amplicons for deep sequencing. We applied nMETR for human DNA using BspFNI enzyme and retrotransposon Alu-specific primers. 454-sequencing enabled identification of 1113 nMETR tag sites, of them ~65% were parts of CpG islands. Representation of reads inversely correlated with methylation levels, thus confirming nMETR fidelity. We created software that eliminates background reads and enables to map and annotate individual tags on human genome. nMETR tags may serve as the controls for large-scale epigenetic studies and for identifying unmethylated transposable elements located close to genomic CpG islands.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 16/10 Miklukho-Maklaya, Moscow 117997, Russia.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Baskaev
authors.forename: Andrew
authors.initials: A
authors.lastname: Garazha
authors.forename: Nurshat
authors.initials: N
authors.lastname: Gaifullin
authors.forename: Maria V
authors.initials: MV
authors.lastname: Suntsova
authors.forename: Anastassia A
authors.initials: AA
authors.lastname: Zabolotneva
authors.forename: Anton A
authors.initials: AA
authors.lastname: Buzdin
mesh_terms: D020087:Alu Elements; D001483:Base Sequence; D018899:CpG Islands; D019175:DNA Methylation / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes; D005821:Genetic Techniques / Q000706:statistics & numerical data; D015894:Genome, Human; D006801:Humans; D017422:Sequence Analysis, DNA; D016324:Sequence Tagged Sites; D012984:Software
chemical_list: D017931:DNA Primers; D004262:DNA Restriction Enzymes