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      Direct detection of DNA methylation during single-molecule, real-time sequencing

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          Abstract

          We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenosine, 5-methylcytosine, and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of the primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We utilize these kinetic signatures to identify adenosine methylation in genomic samples and show that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns within even highly repetitive genomic regions.

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          Most cited references 36

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          Computational Many-Particle Physics

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            Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.

            DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference-mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.
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              Human DNA methylomes at base resolution show widespread epigenomic differences.

              DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA-protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nature methods
                1548-7091
                1548-7105
                28 April 2010
                9 May 2010
                June 2010
                1 December 2010
                : 7
                : 6
                : 461-465
                Affiliations
                [1 ]Pacific Biosciences, 1505 Adams Drive, Menlo Park, CA 94025
                Author notes
                Correspondence should be addressed to S.W.T. ( sturner@ 123456pacificbiosciences.com )
                Article
                nihpa196783
                10.1038/nmeth.1459
                2879396
                20453866

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Funding
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: RC2 HG005618-01 ||HG
                Categories
                Article

                Life sciences

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