关键词: Alu DNA methylation ERV Endogenous retrovirus HT-TREBS LINE Long interspersed element Long terminal repeat Repeat elements Retrotransposons SINE Short interspersed element

Mesh : DNA Methylation Epigenomics / methods Genetic Loci High-Throughput Nucleotide Sequencing / methods Humans Retroelements Sequence Analysis, DNA / methods Sulfites

来  源:   DOI:10.1007/978-1-4939-9004-7_15   PDF(Sci-hub)

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.
摘要:
高通量靶向重复元件亚硫酸氢盐测序(HT-TREBS)旨在测定靶向家族的单个反转录转座子基因座的甲基化水平,以特定于基因座的方式,在全基因组范围内。简而言之,基因组DNA被剪切并连接到离子激流A衔接子(与甲基化的胞嘧啶),其次是亚硫酸氢盐转化,并使用设计用于结合靶向反转录转座子的引物进行扩增。由于引物携带IonTorrentP1衔接子作为5'延伸,扩增的文库已准备好在下一代测序平台上进行大小选择和测序。一旦测序,每个反转录转座子都被定位到一个特定的基因组位点,这是通过确保与侧翼独特序列至少10-bp的重叠来实现的,然后使用BiQ分析仪HT计算映射的反转录转座子的甲基化水平。本章描述了用于文库构建以及HT-TREBS的生物信息学的完整方案。
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