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Analysis of Somatic LINE-1 Insertions in Neurons

  • Francisco J. Sanchez-Luque
  • Sandra R. Richardson
  • Geoffrey J. FaulknerEmail author
Protocol
Part of the Neuromethods book series (NM, volume 131)

Abstract

The method described here is designed to detect and localize somatic genome variation caused by the human retrotransposon LINE-1 (L1) in the genome of neuronal cells. This method combines single-cell manipulation and whole genome amplification technology with a hybridization-based, high-throughput sequencing method called Retrotransposon Capture sequencing (RC-seq) for the precise analysis of the L1 insertion content of single cell genomes. The method is divided into four major sections: extraction of neuronal nuclei and single nuclei isolation; whole genome amplification; RC-seq; and experimental validation of putative insertions.

Key words

Retrotransposition Somatic mosaicism Single-cell Whole genome sequencing (WGS) LINE-1 Mobile genetic element Neurogenesis 

Notes

Acknowledgments

G.J.F. acknowledges the support of a CSL Centenary Fellowship. F.J.S-L. was supported by a postdoctoral fellowship from the Alfonso Martín Escudero Foundation (Spain) and the Peoples Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement No PIOF-GA-2013-623324.

We also acknowledge the significant contribution of Dr. Adam D. Ewing in TEBreak settings adjustment for RC-seq sequencing data analysis, and J. Samuel Jesuadian and Marie-Jeanne H. C. Kempen in technical assistance.

References

  1. 1.
    Lander ES, Linton LM, Birren B et al (2001) Initial sequencing and analysis of the human genome. Nature 409(6822):860–921. doi: 10.1038/35057062 CrossRefPubMedGoogle Scholar
  2. 2.
    Lindblad-Toh K, Wade CM, Mikkelsen TS et al (2005) Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 438(7069):803–819. doi: 10.1038/nature04338 CrossRefPubMedGoogle Scholar
  3. 3.
    Mouse Genome Sequencing C, Waterston RH, Lindblad-Toh K et al (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420(6915):520–562. doi: 10.1038/nature01262 CrossRefGoogle Scholar
  4. 4.
    Craig NL, Caraigie R, Gellert M et al (2002) Mobile DNA II. ASM Press, Washington, DCCrossRefGoogle Scholar
  5. 5.
    Boeke JD, Garfinkel DJ, Styles CA et al (1985) Ty elements transpose through an RNA intermediate. Cell 40(3):491–500CrossRefPubMedGoogle Scholar
  6. 6.
    Luan DD, Korman MH, Jakubczak JL et al (1993) Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72(4):595–605CrossRefPubMedGoogle Scholar
  7. 7.
    Batzer MA, Deininger PL (2002) Alu repeats and human genomic diversity. Nat Rev Genet 3(5):370–379. doi: 10.1038/nrg798 CrossRefPubMedGoogle Scholar
  8. 8.
    Hancks DC, Kazazian HH Jr (2010) SVA retrotransposons: evolution and genetic instability. Semin Cancer Biol 20(4):234–245. doi: 10.1016/j.semcancer.2010.04.001 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Khan H, Smit A, Boissinot S (2006) Molecular evolution and tempo of amplification of human LINE-1 retrotransposons since the origin of primates. Genome Res 16(1):78–87. doi: 10.1101/gr.4001406 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Smit AF, Toth G, Riggs AD et al (1995) Ancestral, mammalian-wide subfamilies of LINE-1 repetitive sequences. J Mol Biol 246(3):401–417. doi: 10.1006/jmbi.1994.0095 CrossRefPubMedGoogle Scholar
  11. 11.
    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):e1002384. doi: 10.1371/journal.pgen.1002384 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Grimaldi G, Skowronski J, Singer MF (1984) Defining the beginning and end of KpnI family segments. EMBO J 3(8):1753–1759PubMedPubMedCentralGoogle Scholar
  13. 13.
    Dombroski BA, Mathias SL, Nanthakumar E et al (1991) Isolation of an active human transposable element. Science 254(5039):1805–1808CrossRefPubMedGoogle Scholar
  14. 14.
    Swergold GD (1990) Identification, characterization, and cell specificity of a human LINE-1 promoter. Mol Cell Biol 10(12):6718–6729CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Garcia-Perez JL, Doucet AJ, Bucheton A et al (2007) Distinct mechanisms for trans-mediated mobilization of cellular RNAs by the LINE-1 reverse transcriptase. Genome Res 17(5):602–611. doi: 10.1101/gr.5870107 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Gilbert N, Lutz S, Morrish TA et al (2005) Multiple fates of L1 retrotransposition intermediates in cultured human cells. Mol Cell Biol 25(17):7780–7795. doi: 10.1128/MCB.25.17.7780-7795.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Richardson SR, Salvador-Palomeque C, Faulkner GJ (2014) Diversity through duplication: whole-genome sequencing reveals novel gene retrocopies in the human population. BioEssays 36(5):475–481. doi: 10.1002/bies.201300181 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Esnault C, Maestre J, Heidmann T (2000) Human LINE retrotransposons generate processed pseudogenes. Nat Genet 24(4):363–367. doi: 10.1038/74184 CrossRefPubMedGoogle Scholar
  19. 19.
    Dewannieux M, Esnault C, Heidmann T (2003) LINE-mediated retrotransposition of marked Alu sequences. Nat Genet 35(1):41–48. doi: 10.1038/ng1223 CrossRefPubMedGoogle Scholar
  20. 20.
    Hancks DC, Goodier JL, Mandal PK et al (2011) Retrotransposition of marked SVA elements by human L1s in cultured cells. Hum Mol Genet 20(17):3386–3400. doi: 10.1093/hmg/ddr245 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Cost GJ, Feng Q, Jacquier A et al (2002) Human L1 element target-primed reverse transcription in vitro. EMBO J 21(21):5899–5910CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Jurka J (1997) Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons. Proc Natl Acad Sci U S A 94(5):1872–1877CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Venter JC, Adams MD, Myers EW et al (2001) The sequence of the human genome. Science 291(5507):1304–1351. doi: 10.1126/science.1058040 CrossRefPubMedGoogle Scholar
  24. 24.
    Kazazian HH Jr, Wong C, Youssoufian H et al (1988) Haemophilia a resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature 332(6160):164–166. doi: 10.1038/332164a0 CrossRefPubMedGoogle Scholar
  25. 25.
    Hancks DC, Kazazian HH Jr (2012) Active human retrotransposons: variation and disease. Curr Opin Genet Dev 22(3):191–203. doi: 10.1016/j.gde.2012.02.006 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hancks DC, Kazazian HH Jr (2016) Roles for retrotransposon insertions in human disease. Mob DNA 7:9. doi: 10.1186/s13100-016-0065-9 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Beck CR, Collier P, Macfarlane C et al (2010) LINE-1 retrotransposition activity in human genomes. Cell 141(7):1159–1170. doi: 10.1016/j.cell.2010.05.021 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Brouha B, Schustak J, Badge RM et al (2003) Hot L1s account for the bulk of retrotransposition in the human population. Proc Natl Acad Sci U S A 100(9):5280–5285. doi: 10.1073/pnas.0831042100 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Philippe C, Vargas-Landin DB, Doucet AJ et al (2016) Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci. elife 5. doi: 10.7554/eLife.13926
  30. 30.
    Sassaman DM, Dombroski BA, Moran JV et al (1997) Many human L1 elements are capable of retrotransposition. Nat Genet 16(1):37–43. doi: 10.1038/ng0597-37 CrossRefPubMedGoogle Scholar
  31. 31.
    Moran JV, Holmes SE, Naas TP et al (1996) High frequency retrotransposition in cultured mammalian cells. Cell 87(5):917–927CrossRefPubMedGoogle Scholar
  32. 32.
    Coufal NG, Garcia-Perez JL, Peng GE et al (2009) L1 retrotransposition in human neural progenitor cells. Nature 460(7259):1127–1131. doi: 10.1038/nature08248 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Kuwabara T, Hsieh J, Muotri A et al (2009) Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis. Nat Neurosci 12(9):1097–1105. doi: 10.1038/nn.2360 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Muotri AR, Chu VT, Marchetto MC et al (2005) Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition. Nature 435(7044):903–910. doi: 10.1038/nature03663 CrossRefPubMedGoogle Scholar
  35. 35.
    Muotri AR, Marchetto MC, Coufal NG et al (2010) L1 retrotransposition in neurons is modulated by MeCP2. Nature 468(7322):443–446. doi: 10.1038/nature09544 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Richardson SR, Morell S, Faulkner GJ (2014) L1 retrotransposons and somatic mosaicism in the brain. Annu Rev Genet 48:1–27. doi: 10.1146/annurev-genet-120213-092412 CrossRefPubMedGoogle Scholar
  37. 37.
    Hozumi N, Tonegawa S (1976) Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc Natl Acad Sci U S A 73(10):3628–3632CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Muramatsu M, Kinoshita K, Fagarasan S et al (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102(5):553–563CrossRefPubMedGoogle Scholar
  39. 39.
    Beck CR, Garcia-Perez JL, Badge RM et al (2011) LINE-1 elements in structural variation and disease. Annu Rev Genomics Hum Genet 12:187–215. doi: 10.1146/annurev-genom-082509-141802 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Hulme AE, Kulpa DA, Garcia-Perez JL et al (2006) The impact of LINE-1 retrotransposition on the human genome. In: Lupski JS (ed) Genomic disorders: the genomic basis of disease. Humana Press, Totowa, NJ, pp 35–72CrossRefGoogle Scholar
  41. 41.
    Baillie JK, Barnett MW, Upton KR et al (2011) Somatic retrotransposition alters the genetic landscape of the human brain. Nature 479(7374):534–537. doi: 10.1038/nature10531 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Shukla R, Upton KR, Munoz-Lopez M et al (2013) Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell 153(1):101–111. doi: 10.1016/j.cell.2013.02.032 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Badge RM, Alisch RS, Moran JV (2003) ATLAS: a system to selectively identify human-specific L1 insertions. Am J Hum Genet 72(4):823–838. doi: 10.1086/373939 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Erwin JA, Paquola AC, Singer T et al (2016) L1-associated genomic regions are deleted in somatic cells of the healthy human brain. Nat Neurosci 19(12):1583–1591. doi: 10.1038/n.4388
  45. 45.
    Ewing AD, Kazazian HH Jr (2010) High-throughput sequencing reveals extensive variation in human-specific L1 content in individual human genomes. Genome Res 20(9):1262–1270. doi: 10.1101/gr.106419.110 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Witherspoon DJ, Xing J, Zhang Y et al (2010) Mobile element scanning (ME-scan) by targeted high-throughput sequencing. BMC Genomics 11:410. doi: 10.1186/1471-2164-11-410 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Evrony GD, Cai X, Lee E et al (2012) Single-neuron sequencing analysis of L1 retrotransposition and somatic mutation in the human brain. Cell 151(3):483–496. doi: 10.1016/j.cell.2012.09.035 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Evrony GD, Lee E, Mehta BK et al (2015) Cell lineage analysis in human brain using endogenous retroelements. Neuron 85(1):49–59. doi: 10.1016/j.neuron.2014.12.028 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Upton KR, Gerhardt DJ, Jesuadian JS et al (2015) Ubiquitous L1 mosaicism in hippocampal neurons. Cell 161(2):228–239. doi: 10.1016/j.cell.2015.03.026 CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Sanchez-Luque FJ, Richardson SR, Faulkner GJ (2016) Retrotransposon capture sequencing (RC-Seq): a targeted, high-throughput approach to resolve somatic L1 retrotransposition in humans. Methods Mol Biol 1400:47–77. doi: 10.1007/978-1-4939-3372-3_4 CrossRefPubMedGoogle Scholar
  51. 51.
    Jiang Y, Matevossian A, Huang HS et al (2008) Isolation of neuronal chromatin from brain tissue. BMC Neurosci 9:42. doi: 10.1186/1471-2202-9-42 CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Okada S, Saiwai H, Kumamaru H et al (2011) Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue. J Cell Physiol 226(2):552–558. doi: 10.1002/jcp.22365 CrossRefPubMedGoogle Scholar
  53. 53.
    Dean FB, Hosono S, Fang L et al (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci U S A 99(8):5261–5266. doi: 10.1073/pnas.082089499 CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Zong C, Lu S, Chapman AR et al (2012) Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338(6114):1622–1626. doi: 10.1126/science.1229164 CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Carreira PE, Ewing AD, Li G et al (2016) Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme. Mob DNA 7:21. doi: 10.1186/s13100-016-0076-6 CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Klawitter S, Fuchs NV, Upton KR et al (2016) Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells. Nat Commun 7:10286. doi: 10.1038/ncomms10286 CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Streva VA, Jordan VE, Linker S et al (2015) Sequencing, identification and mapping of primed L1 elements (SIMPLE) reveals significant variation in full length L1 elements between individuals. BMC Genomics 16:220. doi: 10.1186/s12864-015-1374-y CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Li H (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. http://arxiv.org/abs/1303.3997.
  59. 59.
    Langmead B, Salzberg SL (2012) Fast gapped-read alignment with bowtie 2. Nat Methods 9(4):357–359. doi: 10.1038/nmeth.1923 CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Rahbari R, Badge RM (2016) Combining amplification typing of L1 active subfamilies (ATLAS) with high-throughput sequencing. Methods Mol Biol 1400:95–106. doi: 10.1007/978-1-4939-3372-3_6 CrossRefPubMedGoogle Scholar
  61. 61.
    Scott EC, Gardner EJ, Masood A et al (2016) A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer. Genome Res 26(6):745–755. doi: 10.1101/gr.201814.115 CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Gilbert N, Lutz-Prigge S, Moran JV (2002) Genomic deletions created upon LINE-1 retrotransposition. Cell 110(3):315–325CrossRefPubMedGoogle Scholar
  63. 63.
    Morrish TA, Gilbert N, Myers JS et al (2002) DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat Genet 31(2):159–165. doi: 10.1038/ng898 CrossRefPubMedGoogle Scholar
  64. 64.
    Jurka J (1998) Repeats in genomic DNA: mining and meaning. Curr Opin Struct Biol 8(3):333–337CrossRefPubMedGoogle Scholar
  65. 65.
    Boissinot S, Chevret P, Furano AV (2000) L1 (LINE-1) retrotransposon evolution and amplification in recent human history. Mol Biol Evol 17(6):915–928CrossRefPubMedGoogle Scholar
  66. 66.
    Ovchinnikov I, Rubin A, Swergold GD (2002) Tracing the LINEs of human evolution. Proc Natl Acad Sci U S A 99(16):10522–10527. doi: 10.1073/pnas.152346799 CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Skerra A (1992) Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Res 20(14):3551–3554CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Ostertag EM, Kazazian HH Jr (2001) Twin priming: a proposed mechanism for the creation of inversions in L1 retrotransposition. Genome Res 11(12):2059–2065. doi: 10.1101/gr.205701 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Francisco J. Sanchez-Luque
    • 1
    • 2
  • Sandra R. Richardson
    • 1
  • Geoffrey J. Faulkner
    • 1
    • 3
    Email author
  1. 1.Mater Research Institute—University of QueenslandWoolloongabbaAustralia
  2. 2.Pfizer-Andalusian Government-University of Granada Centre for Genomics and Oncologic Research (Genyo)GranadaSpain
  3. 3.Queensland Brain InstituteUniversity of QueenslandBrisbaneAustralia

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