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bioRxiv · 10.1101/163535

HAM-TBS: High accuracy methylation measurements via targeted bisulfite sequencing

Roeh, S.·Provencal, N.·Wiechmann, T.·Koedel, M.·Sauer, S.·Binder, E. B.
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Abstract

BackgroundThe ability to accurately and efficiently measure DNA methylation is vital to advance the understanding of this mechanism and its contribution to common diseases. Here, we present a highly accurate method to measure methylation using bisulfite sequencing (termed HAM-TBS). This novel method is able to assess DNA methylation in multiple samples with high accuracy in a cost-effective manner. We developed this assay for the FKBP5 locus, an important gene in the regulation of the stress system and previously linked to stress-related disorders, but the method is applicable to any locus of interest.\n\nResultsHAM-TBS enables multiplexing of up to 96 samples spanning a region of ~10 kb using the llumina MiSeq. It incorporates a triplicate bisulfite conversion step, pooled target enrichment via PCR, PCR-free library preparation and a minimum coverage of 1,000x. Furthermore, we designed and validated a targeted panel to specifically assess regulatory regions within the FKBP5 locus including the transcription start site, topologically associated domain boundaries, intergenic and proximal enhancers as well as glucocorticoid receptor and CTCF binding sites that are not covered in commercially available DNA methylation arrays.\n\nConclusionsHAM-TBS represents a highly accurate, medium-throughput sequencing approach for robust detection of DNA methylation changes in specific target regions.

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Roeh, S., Provencal, N., Wiechmann, T., Koedel, M., Sauer, S., Binder, E. B.. 2017-07-14. HAM-TBS: High accuracy methylation measurements via targeted bisulfite sequencing. https://doi.org/10.1101/163535

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