Title
High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS)
Document Type
Article
Publication Date
1-1-2019
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.
Publication Source (Journal or Book title)
Methods in molecular biology (Clifton, N.J.)
First Page
219
Last Page
228
Recommended Citation
Bakshi, A., Ekram, M. B., & Kim, J. (2019). High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS). Methods in molecular biology (Clifton, N.J.), 1908, 219-228. https://doi.org/10.1007/978-1-4939-9004-7_15