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Methods

Decoding the fine-scale structure of a breast cancer genome and transcriptome

¹ Department of Urology, and Cancer Research Institute, University of California San Francisco Comprehensive Cancer Center, San Francisco, California 94115, USA ² Department of Computer Science & Engineering, University of California, San Diego, La Jolla, California 92093, USA ³ The Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, United Kingdom ⁴ Department of Radiation Oncology, MCB 200, San Francisco, California 94103, USA ⁵ Amplicon Express, Pullman, Washington 99163, USA ⁶ Lawrence Berkeley National Laboratory, Life Sciences Division and MS 84R171 Berkeley, California 94720, USA ⁷ Genomics Division and Joint Genome Institute, MS 84R171 Berkeley, California 94720, USA ⁸ Evrogen JSC, Miklukho-Maklaya 16/10, Moscow, Russia 117997 ⁹ BACPAC Resources Children's Hospital Oakland, Oakland, California 94609, USA

A comprehensive understanding of cancer is predicated upon knowledge of the structure of malignant genomes underlying its many variant forms and the molecular mechanisms giving rise to them. It is well established that solid tumor genomes accumulate a large number of genome rearrangements during tumorigenesis. End Sequence Profiling (ESP) maps and clones genome breakpoints associated with all types of genome rearrangements elucidating the structural organization of tumor genomes. Here we extend the ESP methodology in several directions using the breast cancer cell line MCF-7. First, targeted ESP is applied to multiple amplified loci, revealing a complex process of rearrangement and coamplification in these regions reminiscent of breakage/fusion/bridge cycles. Second, genome breakpoints identified by ESP are confirmed using a combination of DNA sequencing and PCR. Third, in vitro functional studies assign biological function to a rearranged tumor BAC clone, demonstrating that it encodes antiapoptotic activity. Finally, ESP is extended to the transcriptome identifying four novel fusion transcripts and providing evidence that expression of fusion genes may be common in tumors. These results demonstrate the distinct advantages of ESP including: (1) the ability to detect all types of rearrangements and copy number changes; (2) straightforward integration of ESP data with the annotated genome sequence; (3) immortalization of the genome; (4) ability to generate tumor-specific reagents for in vitro and in vivo functional studies. Given these properties, ESP could play an important role in a tumor genome project.

Article published online ahead of print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.4247306.

¹⁰ Corresponding author.
E-mail collins{at}cc.ucsf.edu; fax (415) 476-8218.

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