Abstract
High-throughput targeted repeat element bisulfite sequencing (HT-TREBS) is designed to assay the methylation level of individual retrotransposon loci of a targeted family, in a locus-specific manner, and on a genome-wide scale. Briefly, genomic DNA is sheared and ligated to Ion Torrent A adaptors (with methylated cytosines), followed by bisulfite-conversion, and amplification with primers designed to bind the targeted retrotransposon. Since the primers carry the Ion Torrent P1 adaptor as a 5′-extension, the amplified library is ready to be size-selected and sequenced on a next-generation sequencing platform. Once sequenced, each retrotransposon is mapped to a particular genomic locus, which is achieved through ensuring at least a 10-bp overlap with flanking unique sequence, followed by the calculation of methylation levels of the mapped retrotransposon using a BiQ Analyzer HT. A complete protocol for library construction as well as the bioinformatics for HT-TREBS is described in this chapter.
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Bakshi, A., Ekram, M.B., Kim, J. (2019). High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS). In: Murray, S. (eds) Tumor Profiling. Methods in Molecular Biology, vol 1908. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9004-7_15
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DOI: https://doi.org/10.1007/978-1-4939-9004-7_15
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