Skip to main content
SpringerLink
Log in
Book cover

Genomic Mosaicism in Neurons and Other Cell Types pp 109–131Cite as

Single-Cell CNV Detection in Human Neuronal Nuclei

  • Protocol
  • First Online:

Part of the book series: Neuromethods ((NM,volume 131))

Abstract

Genomic mosaicism is prevalent throughout human somatic tissues and is much more common than previously thought. Here, we describe step-by-step methods to isolate neuronal nuclei from human brain and identify megabase-scale copy number variants (CNVs) in single nuclei. The approach detailed herein includes use of CellRaft technology for single-nucleus isolation, the PicoPLEX approach to whole-genome amplification and library preparation, and a pooled library purification protocol, termed Gel2Gel, which has been developed in our laboratory. These methods are focused toward neuroscience research, but are adaptable to many biomedical fields.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Stern C (1931) Analyse eines Mosaikindividuums bei Drosophila melanogaster. Bio Zentr 51:194–199

    Google Scholar 

  2. Stern C (1936) Somatic crossing over and segregation in Drosophila Melanogaster. Genetics 21(6):625–730

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Stern C, Doan D (1936) A cytogenetic demonstration of crossing-over between X- and Y- chromosomes in the male Drosophila Melanogaster. Proc Natl Acad Sci U S A 22(11):649–654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Stern C, Rentschler V (1936) The effect of temperature on the frequency of somatic crossing-over in Drosophila Melanogaster. Proc Natl Acad Sci U S A 22(7):451–453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cotterman CW (1956) Somatic mosaicism for antigen A2. Acta Genet Stat Med 6(4):520–521

    PubMed  Google Scholar 

  6. Levan A (1956) Chromosome studies on some human tumors and tissues of normal origin, grown in vivo and in vitro at the Sloan-Kettering institute. Cancer 9(4):648–663

    Article  CAS  PubMed  Google Scholar 

  7. Campbell IM, Shaw CA, Stankiewicz P, Lupski JR (2015) Somatic mosaicism: implications for disease and transmission genetics. Trends Genet 31(7):382–392. doi:10.1016/j.tig.2015.03.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Campbell IM, Yuan B, Robberecht C, Pfundt R, Szafranski P, McEntagart ME, Nagamani SC, Erez A, Bartnik M, Wisniowiecka-Kowalnik B, Plunkett KS, Pursley AN, Kang SH, Bi W, Lalani SR, Bacino CA, Vast M, Marks K, Patton M, Olofsson P, Patel A, Veltman JA, Cheung SW, Shaw CA, Vissers LE, Vermeesch JR, Lupski JR, Stankiewicz P (2014) Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders. Am J Hum Genet 95(2):173–182. doi:10.1016/j.ajhg.2014.07.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jacobs KB, Yeager M, Zhou W, Wacholder S, Wang Z, Rodriguez-Santiago B, Hutchinson A, Deng X, Liu C, Horner MJ, Cullen M, Epstein CG, Burdett L, Dean MC, Chatterjee N, Sampson J, Chung CC, Kovaks J, Gapstur SM, Stevens VL, Teras LT, Gaudet MM, Albanes D, Weinstein SJ, Virtamo J, Taylor PR, Freedman ND, Abnet CC, Goldstein AM, Hu N, Yu K, Yuan JM, Liao L, Ding T, Qiao YL, Gao YT, Koh WP, Xiang YB, Tang ZZ, Fan JH, Aldrich MC, Amos C, Blot WJ, Bock CH, Gillanders EM, Harris CC, Haiman CA, Henderson BE, Kolonel LN, Le Marchand L, McNeill LH, Rybicki BA, Schwartz AG, Signorello LB, Spitz MR, Wiencke JK, Wrensch M, Wu X, Zanetti KA, Ziegler RG, Figueroa JD, Garcia-Closas M, Malats N, Marenne G, Prokunina-Olsson L, Baris D, Schwenn M, Johnson A, Landi MT, Goldin L, Consonni D, Bertazzi PA, Rotunno M, Rajaraman P, Andersson U, Beane Freeman LE, Berg CD, Buring JE, Butler MA, Carreon T, Feychting M, Ahlbom A, Gaziano JM, Giles GG, Hallmans G, Hankinson SE, Hartge P, Henriksson R, Inskip PD, Johansen C, Landgren A, McKean-Cowdin R, Michaud DS, Melin BS, Peters U, Ruder AM, Sesso HD, Severi G, Shu XO, Visvanathan K, White E, Wolk A, Zeleniuch-Jacquotte A, Zheng W, Silverman DT, Kogevinas M, Gonzalez JR, Villa O, Li D, Duell EJ, Risch HA, Olson SH, Kooperberg C, Wolpin BM, Jiao L, Hassan M, Wheeler W, Arslan AA, Bueno-de-Mesquita HB, Fuchs CS, Gallinger S, Gross MD, Holly EA, Klein AP, LaCroix A, Mandelson MT, Petersen G, Boutron-Ruault MC, Bracci PM, Canzian F, Chang K, Cotterchio M, Giovannucci EL, Goggins M, Hoffman Bolton JA, Jenab M, Khaw KT, Krogh V, Kurtz RC, McWilliams RR, Mendelsohn JB, Rabe KG, Riboli E, Tjonneland A, Tobias GS, Trichopoulos D, Elena JW, Yu H, Amundadottir L, Stolzenberg-Solomon RZ, Kraft P, Schumacher F, Stram D, Savage SA, Mirabello L, Andrulis IL, Wunder JS, Patino Garcia A, Sierrasesumaga L, Barkauskas DA, Gorlick RG, Purdue M, Chow WH, Moore LE, Schwartz KL, Davis FG, Hsing AW, Berndt SI, Black A, Wentzensen N, Brinton LA, Lissowska J, Peplonska B, McGlynn KA, Cook MB, Graubard BI, Kratz CP, Greene MH, Erickson RL, Hunter DJ, Thomas G, Hoover RN, Real FX, Fraumeni JF Jr, Caporaso NE, Tucker M, Rothman N, Perez-Jurado LA, Chanock SJ (2012) Detectable clonal mosaicism and its relationship to aging and cancer. Nat Genet 44(6):651–658. doi:10.1038/ng.2270

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lupski JR (2013) Genetics. Genome mosaicism-one human, multiple genomes. Science 341(6144):358–359. doi:10.1126/science.1239503

    Article  CAS  PubMed  Google Scholar 

  11. Stern C (1968) Genetic mosaics in animals and man. In: Genetic mosaics and other essays. Harvard University Press, Cambridge, MA

    Chapter  Google Scholar 

  12. De S (2011) Somatic mosaicism in healthy human tissues. Trends Genet 27(6):217–223. doi:10.1016/j.tig.2011.03.002

    Article  CAS  PubMed  Google Scholar 

  13. Gottlieb B, Beitel LK, Trifiro MA (2001) Somatic mosaicism and variable expressivity. Trends Genet 17(2):79–82

    Article  CAS  PubMed  Google Scholar 

  14. Hall JG (1988) Review and hypotheses: somatic mosaicism: observations related to clinical genetics. Am J Hum Genet 43(4):355–363

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Mkrtchyan H, Gross M, Hinreiner S, Polytiko A, Manvelyan M, Mrasek K, Kosyakova N, Ewers E, Nelle H, Liehr T, Bhatt S, Thoma K, Gebhart E, Wilhelm S, Fahsold R, Volleth M, Weise A (2010) The human genome puzzle—the role of copy number variation in somatic mosaicism. Curr Genomics 11(6):426–431. doi:10.2174/138920210793176047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Taylor TH, Gitlin SA, Patrick JL, Crain JL, Wilson JM, Griffin DK (2014) The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans. Hum Reprod Update 20(4):571–581. doi:10.1093/humupd/dmu016

    Article  CAS  PubMed  Google Scholar 

  17. Thibodeau IL, Xu J, Li Q, Liu G, Lam K, Veinot JP, Birnie DH, Jones DL, Krahn AD, Lemery R, Nicholson BJ, Gollob MH (2010) Paradigm of genetic mosaicism and lone atrial fibrillation: physiological characterization of a connexin 43-deletion mutant identified from atrial tissue. Circulation 122(3):236–244. doi:10.1161/CIRCULATIONAHA.110.961227

    Article  CAS  PubMed  Google Scholar 

  18. Vig BK (1978) Somatic mosaicism in plants with special reference to somatic crossing over. Environ Health Perspect 27:27–36

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Youssoufian H, Pyeritz RE (2002) Mechanisms and consequences of somatic mosaicism in humans. Nat Rev Genet 3(10):748–758. doi:10.1038/nrg906

    Article  CAS  PubMed  Google Scholar 

  20. Insel TR (2014) Brain somatic mutations: the dark matter of psychiatric genetics? Mol Psychiatry 19(2):156–158. doi:10.1038/mp.2013.168

    Article  CAS  PubMed  Google Scholar 

  21. Erwin JA, Paquola AC, Singer T, Gallina I, Novotny M, Quayle C, Bedrosian TA, Alves FI, Butcher CR, Herdy JR, Sarkar A, Lasken RS, Muotri AR, Gage FH (2016) L1-associated genomic regions are deleted in somatic cells of the healthy human brain. Nat Neurosci 19(12):1583–1591. doi:10.1038/nn.4388

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lodato MA, Woodworth MB, Lee S, Evrony GD, Mehta BK, Karger A, Lee S, Chittenden TW, D’Gama AM, Cai X, Luquette LJ, Lee E, Park PJ, Walsh CA (2015) Somatic mutation in single human neurons tracks developmental and transcriptional history. Science 350(6256):94–98. doi:10.1126/science.aab1785

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cai X, Evrony GD, Lehmann HS, Elhosary PC, Mehta BK, Poduri A, Walsh CA (2014) Single cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain. Cell Rep 8(5):1280–1289. doi:10.1016/j.celrep.2014.07.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. McConnell MJ, Lindberg MR, Brennand KJ, Piper JC, Voet T, Cowing-Zitron C, Shumilina S, Lasken RS, Vermeesch JR, Hall IM, Gage FH (2013) Mosaic copy number variation in human neurons. Science 342(6158):632–637. doi:10.1126/science.1243472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Knouse KA, Wu J, Amon A (2016) Assessment of megabase-scale somatic copy number variation using single cell sequencing. Genome Res 26(3):376–384. doi:10.1101/gr.198937.115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hawkins TL, Detter JC, Richardson PM (2002) Whole genome amplification-applications and advances. Curr Opin Biotechnol 13(1):65–67

    Article  CAS  PubMed  Google Scholar 

  27. Hou Y, Wu K, Shi X, Li F, Song L, Wu H, Dean M, Li G, Tsang S, Jiang R, Zhang X, Li B, Liu G, Bedekar N, Lu N, Xie G, Liang H, Chang L, Wang T, Chen J, Li Y, Zhang X, Yang H, Xu X, Wang L, Wang J (2015) Comparison of variations detection between whole genome amplification methods used in single cell resequencing. Gigascience 4:37. doi:10.1186/s13742-015-0068-3

    Article  PubMed  PubMed Central  Google Scholar 

  28. Han T, Chang CW, Kwekel JC, Chen Y, Ge Y, Martinez-Murillo F, Roscoe D, Tezak Z, Philip R, Bijwaard K, Fuscoe JC (2012) Characterization of whole genome amplified (WGA) DNA for use in genotyping assay development. BMC Genomics 13:217. doi:10.1186/1471-2164-13-217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pugh TJ, Delaney AD, Farnoud N, Flibotte S, Griffith M, Li HI, Qian H, Farinha P, Gascoyne RD, Marra MA (2008) Impact of whole genome amplification on analysis of copy number variants. Nucleic Acids Res 36(13):e80. doi:10.1093/nar/gkn378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Insel TR, Gogtay N (2014) National Institute of Mental Health clinical trials: new opportunities, new expectations. JAMA Psychiat 71(7):745–746. doi:10.1001/jamapsychiatry.2014.426

    Article  Google Scholar 

  31. Insel TR (2014) The NIMH research domain criteria (RDoC) project: precision medicine for psychiatry. Am J Psychiatry 171(4):395–397. doi:10.1176/appi.ajp.2014.14020138

    Article  PubMed  Google Scholar 

  32. Chung JY, Insel TR (2014) Mind the gap: neuroscience literacy and the next generation of psychiatrists. Acad Psychiatry 38(2):121–123. doi:10.1007/s40596-014-0054-6

    Article  PubMed  Google Scholar 

  33. Damianov A, Black DL (2010) Autoregulation of fox protein expression to produce dominant negative splicing factors. RNA 16(2):405–416. doi:10.1261/rna.1838210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kim KK, Adelstein RS, Kawamoto S (2009) Identification of neuronal nuclei (NeuN) as fox-3, a new member of the fox-1 gene family of splicing factors. J Biol Chem 284(45):31052–31061. doi:10.1074/jbc.M109.052969

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lucas CH, Calvez M, Babu R, Brown A (2014) Altered subcellular localization of the NeuN/Rbfox3 RNA splicing factor in HIV-associated neurocognitive disorders (HAND). Neurosci Lett 558:97–102. doi:10.1016/j.neulet.2013.10.037

    Article  CAS  PubMed  Google Scholar 

  36. Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P, Sun Z, Zong Q, Du Y, Du J, Driscoll M, Song W, Kingsmore SF, Egholm M, Lasken RS (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Baslan T, Kendall J, Rodgers L, Cox H, Riggs M, Stepansky A, Troge J, Ravi K, Esposito D, Lakshmi B, Wigler M, Navin N, Hicks J (2012) Genome-wide copy number analysis of single cells. Nat Protoc 7(6):1024–1041. doi:10.1038/nprot.2012.039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Baslan T, Kendall J, Rodgers L, Cox H, Riggs M, Stepansky A, Troge J, Ravi K, Esposito D, Lakshmi B, Wigler M, Navin N, Hicks J (2016) Corrigendum: genome-wide copy number analysis of single cells. Nat Protoc 11(3):616. doi:10.1038/nprot0316.616b

    Article  CAS  PubMed  Google Scholar 

  39. Navin N, Kendall J, Troge J, Andrews P, Rodgers L, McIndoo J, Cook K, Stepansky A, Levy D, Esposito D, Muthuswamy L, Krasnitz A, McCombie WR, Hicks J, Wigler M (2011) Tumour evolution inferred by single cell sequencing. Nature 472(7341):90–94. doi:10.1038/nature09807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zong C, Lu S, Chapman AR, Xie XS (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. de Bourcy CF, De Vlaminck I, Kanbar JN, Wang J, Gawad C, Quake SR (2014) A quantitative comparison of single cell whole genome amplification methods. PLoS One 9(8):e105585. doi:10.1371/journal.pone.0105585

    Article  PubMed  PubMed Central  Google Scholar 

  42. Gawad C, Koh W, Quake SR (2016) Single cell genome sequencing: current state of the science. Nat Rev Genet 17(3):175–188. doi:10.1038/nrg.2015.16

    Article  CAS  PubMed  Google Scholar 

  43. Blainey PC (2013) The future is now: single cell genomics of bacteria and archaea. FEMS Microbiol Rev 37(3):407–427. doi:10.1111/1574-6976.12015

    Article  CAS  PubMed  Google Scholar 

  44. Li H, Durbin R (2010) Fast and accurate long-read alignment with burrows-Wheeler transform. Bioinformatics 26(5):589–595. doi:10.1093/bioinformatics/btp698

    Article  PubMed  PubMed Central  Google Scholar 

  45. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. doi:10.1093/bioinformatics/btp352

    Article  PubMed  PubMed Central  Google Scholar 

  46. Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26(6):841–842. doi:10.1093/bioinformatics/btq033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Garvin T, Aboukhalil R, Kendall J, Baslan T, Atwal GS, Hicks J, Wigler M, Schatz MC (2015) Interactive analysis and assessment of single cell copy-number variations. Nat Methods 12(11):1058–1060. doi:10.1038/nmeth.3578

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Seshan VE, Olshen A (2016) DNAcopy: DNA copy number data analysis. (R package). 1.48.0. Edn.

    Google Scholar 

  49. Lai D, Ha G, Shah S (2016) HMMcopy: copy number prediction with correction for GC and mappability bias for HTS data (R package). 1.16.0. Edn., Bioconductor

    Google Scholar 

  50. Nilsen G, Liestol K, Van Loo P, Moen Vollan HK, Eide MB, Rueda OM, Chin SF, Russell R, Baumbusch LO, Caldas C, Borresen-Dale AL, Lingjaerde OC (2012) Copynumber: efficient algorithms for single- and multi-track copy number segmentation. BMC Genomics 13:591. doi:10.1186/1471-2164-13-591

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. Margaret B. Wierman, Ian E. Burbulis, and William D. Chronister contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Virginia, School of Medicine, Charlottesville, VA, 22908, USA

    Margaret B. Wierman, Ian E. Burbulis, William D. Chronister, Stefan Bekiranov & Michael J. McConnell

  2. Universidad San Sebastian, Escuela de Medicina, Campus Pichi Pelluco, Lago Panguipulli, 1390, Puerto Montt, Chile

    Ian E. Burbulis

Authors
  1. Margaret B. Wierman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ian E. Burbulis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William D. Chronister
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stefan Bekiranov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael J. McConnell
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael J. McConnell .

Editor information

Editors and Affiliations

  1. Department of Molecular, Cellular and Developmental Neurobiology, Cajal Institute (IC-CSIC), Madrid, Spain

    José María Frade

  2. Biological Studies and Laboratory of Genetics, The Salk Institute, La Jolla, California, USA

    Fred H. Gage

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Wierman, M.B., Burbulis, I.E., Chronister, W.D., Bekiranov, S., McConnell, M.J. (2017). Single-Cell CNV Detection in Human Neuronal Nuclei. In: Frade, J., Gage, F. (eds) Genomic Mosaicism in Neurons and Other Cell Types. Neuromethods, vol 131. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7280-7_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7280-7_6

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7279-1

  • Online ISBN: 978-1-4939-7280-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation