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Home arrow Health arrow DNA Modifications in the Brain. Neuroepigenetic Regulation of Gene Expression
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DISCUSSION

We addressed the problem of uncovering the landscape of DNA methylation of repetitive elements. To this end, we described a unique application of SMRT sequencing to epigenetics. This direction had been already explored in the research community for bacterial and viral species. However, this application in large vertebrate genomes has been largely unexplored because of the subtle cytosine methylation signals in the kinetic information.

Our method uses relatively small amounts of kinetic information by incorporating a model reflecting our prior knowledge on the regional patterns of CpG methylation of vertebrate genomes. We confirmed the validity of our strategy by comparing the prediction to bisulfite sequencing data on medaka and to BeadChip analysis on human samples. These two datasets had very different characteristics, which seemed to be partly because of the methods used and partly because of the nature of the samples used (ie, the medaka samples were derived from an inbred strain, whereas the human samples were from diploid cells). Despite such differences in characteristics, our method using the same parameters performed almost equally well for both datasets. These observations suggested that the choice of parameters is robust for a wide variety of samples, which is a desirable feature for any method.

Our method had important strengths compared with conventional tools for epigenetic studies, such as bisulfite sequencing or affinity-based assays, with not only an expected increase in comprehensiveness by virtue of long SMRT reads but also in the remarkable reduction of laboratory work. If an epigenetic study is conducted alongside a resequencing study or a de novo assembly study using SMRT sequencing, the methylation status could be called solely in silico, and no additional experiments would be necessary.

Methylation analysis of ToI2,a 4682-bp autonomous transposon, in medaka

Figure 7.3 Methylation analysis of ToI2,a 4682-bp autonomous transposon, in medaka. The new genome assembly of single-molecule real-time (SMRT) reads had 17 regions (contigs) that contained complete ToI2 copies. Circles show our prediction of the methylation state of CpG sites, whereas rectangles show the methylation states within each 100-bp window obtained from bisulfite sequencing. For both tracks, red/outlined and blue/filled indicate hypomethylation and hypermethylation, respectively. As the 11th region was located at the extreme of the contig, ToI2 was not observed successfully by either SMRT sequencing or bisulfite sequencing. For the other 16 regions, hypermethylation of ToI2 was observed consistently by SMRT sequencing, whereas virtually no information was available on the ToI2 region from bisulfite sequencing.

 
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