Advertisement

High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS)

  • Arundhati Bakshi
  • Muhammad B. Ekram
  • Joomyeong KimEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1908)

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.

Key words

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

References

  1. 1.
    de Koning AP, Gu W, Castoe TA et al (2011) Repetitive elements may comprise over two-thirds of the human genome. PLoS Genet 7(12):e1002384CrossRefGoogle Scholar
  2. 2.
    Burns KH (2017) Transposable elements in cancer. Nat Rev Cancer 17(7):415–424CrossRefGoogle Scholar
  3. 3.
    Anwar SL, Wulaningsih W, Lehmann U (2017) Transposable elements in human cancer: causes and consequences of deregulation. Int J Mol Sci 18(5):974CrossRefGoogle Scholar
  4. 4.
    Lee E, Iskow R, Yang L et al (2012) Landscape of somatic retrotransposition in human cancers. Science 337(6097):967–971CrossRefGoogle Scholar
  5. 5.
    Penha RCC, Lima SCS, Boroni M et al (2017) Intrinsic LINE-1 hypomethylation and decreased brca1 expression are associated with DNA repair delay in irradiated thyroid cells. Radiat Res 188(2):144–155CrossRefGoogle Scholar
  6. 6.
    Daskalos A, Nikolaidis G, Xinarianos G et al (2009) Hypomethylation of retrotransposable elements correlates with genomic instability in non-small cell lung cancer. Int J Cancer 124(1):81–87CrossRefGoogle Scholar
  7. 7.
    Hur K, Cejas P, Feliu J et al (2014) Hypomethylation of long interspersed nuclear element-1 (LINE-1) leads to activation of proto-oncogenes in human colorectal cancer metastasis. Gut 63(4):635–646CrossRefGoogle Scholar
  8. 8.
    Wolff EM, Byun HM, Han HF et al (2010) Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer. PLoS Genet 6(4):e1000917CrossRefGoogle Scholar
  9. 9.
    Lock FE, Rebollo R, Miceli-Royer K et al (2014) Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma. Proc Natl Sci Acad USA 111(34):E3534–E3543CrossRefGoogle Scholar
  10. 10.
    Park SY, Seo AN, Jung HY et al (2014) Alu and LINE-1 hypomethylation is associated with HER2 enriched subtype of breast cancer. PLoS One 9(6):e100429CrossRefGoogle Scholar
  11. 11.
    Bae JM, Shin SH, Kwon HJ et al (2012) ALU and LINE-1 hypomethylations in multistep gastric carcinogenesis and their prognostic implications. Int J Cancer 131(6):1323–1331CrossRefGoogle Scholar
  12. 12.
    Bollati V, Fabris S, Pegoraro V et al (2009) Differential repetitive DNA methylation in multiple myeloma molecular subgroups. Carcinogenesis 30(8):1330–1335CrossRefGoogle Scholar
  13. 13.
    Akers SN, Moysich K, Zhang W et al (2014) LINE1 and Alu repetitive element DNA methylation in tumors and white blood cells from epithelial ovarian cancer patients. Gynecol Oncol 132(2):462–467CrossRefGoogle Scholar
  14. 14.
    Rhee YY, Lee TH, Song YS et al (2015) Prognostic significance of promoter CpG island hypermethylation and repetitive DNA hypomethylation in stage I lung adenocarcinoma. Virchows Arch 466(6):675–683CrossRefGoogle Scholar
  15. 15.
    Wedge E, Hansen JW (2017) Global hypomethylation is an independent prognostic factor in diffuse large B cell lymphoma. Am J Hematol 92(7):689–694CrossRefGoogle Scholar
  16. 16.
    Swets M, Zaalberg A, Boot A et al (2016) Tumor LINE-1 Methylation Level in Association with Survival of Patients with Stage II Colon Cancer. Int J Mol Sci 18(1):36CrossRefGoogle Scholar
  17. 17.
    Ekram MB, Kim J (2014) High-throughput targeted repeat element bisulfite sequencing (HT-TREBS): genome-wide DNA methylation analysis of IAP LTR retrotransposon. PLoS One 9(7):e101683CrossRefGoogle Scholar
  18. 18.
    Bakshi A, Ekram MB, Kim J (2015) Locus-specific DNA methylation analysis of retrotransposons in ES, somatic and cancer cells using High-Throughput Targeted Repeat Element Bisulfite Sequencing. Genom Data 3:87–89CrossRefGoogle Scholar
  19. 19.
    Clark SJ, Harrison J, Paul CL, Frommer M (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22(15):2990–2997CrossRefGoogle Scholar
  20. 20.
    Bakshi A, Herke SW, Batzer MA, Kim J (2016) DNA methylation variation of human-specific Alu repeats. Epigenetics 11(2):163–173CrossRefGoogle Scholar
  21. 21.
    Pereira FL, Soares SC, Dorella FA et al (2016) Evaluating the efficacy of the new Ion PGM Hi-Q Sequencing Kit applied to bacterial genomes. Genomics 107(5):189–198CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Arundhati Bakshi
    • 1
  • Muhammad B. Ekram
    • 1
  • Joomyeong Kim
    • 1
    Email author
  1. 1.Department of Biological SciencesLouisiana State UniversityBaton RougeUSA

Personalised recommendations