Mapping translocation breakpoints by next-generation sequencing

  1. Wei Chen1,9,
  2. Vera Kalscheuer1,
  3. Andreas Tzschach1,
  4. Corinna Menzel1,
  5. Reinhard Ullmann1,
  6. Marcel Holger Schulz1,2,
  7. Fikret Erdogan1,
  8. Na Li1,
  9. Zofia Kijas1,
  10. Ger Arkesteijn3,
  11. Isidora Lopez Pajares4,
  12. Margret Goetz-Sothmann5,
  13. Uwe Heinrich6,
  14. Imma Rost6,
  15. Andreas Dufke7,
  16. Ute Grasshoff7,
  17. Birgitta Glaeser8,
  18. Martin Vingron1, and
  19. H. Hilger Ropers1
  1. 1 Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany;
  2. 2 International Max-Planck Research School for Computational Biology and Scientific Computing, 14195 Berlin, Germany;
  3. 3 Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3508 TC Utrecht, The Netherlands;
  4. 4 Department of Genetics, Hospital Universitario La Paz, 28046 Madrid, Spain;
  5. 5 Practice of Human Genetics, 81379 Munich, Germany;
  6. 6 Centre of Human Genetics, 82152 Martinsried, Germany;
  7. 7 Department of Medical Genetics, Eberhard Karls University, 72076 Tuebingen, Germany;
  8. 8 Institut für Klinische Genetik, Olgahospital, 70176 Stuttgart, Germany

Abstract

Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterization of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time-consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using “next-generation” (Illumina/Solexa) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows the determination of their exact nucleotide positions within a few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations.

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