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         headline:Clonal evolution in breast cancer revealed by single nucleus genome sequencing
         description:Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER+) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER+ tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer. To investigate genomic diversity within tumours, a new type of whole-genome and exome single cell sequencing has been developed using G2/M nuclei; the technique was used to sequence single nuclei from an oestrogen-positive breast cancer and a triple-negative ductal carcinoma—aneuploidy rearrangements emerged as early events in tumour formation and then point mutations evolved gradually over time. Human breast cancers often display intratumour genomic heterogeneity, making clinical diagnosis difficult and complicating the interpretation of research results. This study tackles the problem using a newly developed whole-genome single-cell sequencing technique called nuc-seq that makes use of the natural genome duplication that occurs in the S phase of the cell cycle to achieve 91% mean coverage breadth. The method is applied to sequence single normal and tumour nuclei from an oestrogen-receptor-positive breast cancer and a triple-negative ductal carcinoma. Aneuploid rearrangements emerge as early events, and they remain stable during clonal expansion. In contrast, point mutations appear to evolve gradually, generating extensive clonal diversity. The data also show that no two single tumour cells are genetically identical, raising interesting questions as to the strict definition of a clone.
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      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
            name:The University of Texas Graduate School of Biomedical Sciences
            address:
               name:The University of Texas Graduate School of Biomedical Sciences, Houston, USA
               type:PostalAddress
            type:Organization
      name:Anna Unruh
      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Whijae Roh
      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Xiuqing Shi
      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Ken Chen
      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Paul Scheet
      affiliation:
            name:The University of Texas Graduate School of Biomedical Sciences
            address:
               name:The University of Texas Graduate School of Biomedical Sciences, Houston, USA
               type:PostalAddress
            type:Organization
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Selina Vattathil
      affiliation:
            name:The University of Texas Graduate School of Biomedical Sciences
            address:
               name:The University of Texas Graduate School of Biomedical Sciences, Houston, USA
               type:PostalAddress
            type:Organization
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Han Liang
      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Asha Multani
      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Hong Zhang
      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      name:Rui Zhao
      affiliation:
            name:Dana-Farber Cancer Institute, Harvard School of Public Health
            address:
               name:Department of Biostatistics and Computational Biology, and Department of Biostatistics, Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, USA
               type:PostalAddress
            type:Organization
      name:Franziska Michor
      affiliation:
            name:Dana-Farber Cancer Institute, Harvard School of Public Health
            address:
               name:Department of Biostatistics and Computational Biology, and Department of Biostatistics, Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, USA
               type:PostalAddress
            type:Organization
      name:Funda Meric-Bernstam
      affiliation:
            name:The University of Texas MD Anderson Cancer Center Department of Investigational Cancer Therapeutics
            address:
               name:The University of Texas MD Anderson Cancer Center Department of Investigational Cancer Therapeutics, Houston, USA
               type:PostalAddress
            type:Organization
      name:Nicholas E. Navin
      affiliation:
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
            name:The University of Texas Graduate School of Biomedical Sciences
            address:
               name:The University of Texas Graduate School of Biomedical Sciences, Houston, USA
               type:PostalAddress
            type:Organization
            name:The University of Texas MD Anderson Cancer Center
            address:
               name:Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
               type:PostalAddress
            type:Organization
      email:[email protected]
PostalAddress:
      name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:The University of Texas Graduate School of Biomedical Sciences, Houston, USA
      name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:The University of Texas Graduate School of Biomedical Sciences, Houston, USA
      name:Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:The University of Texas Graduate School of Biomedical Sciences, Houston, USA
      name:Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:Department of Biostatistics and Computational Biology, and Department of Biostatistics, Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, USA
      name:Department of Biostatistics and Computational Biology, and Department of Biostatistics, Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, USA
      name:The University of Texas MD Anderson Cancer Center Department of Investigational Cancer Therapeutics, Houston, USA
      name:Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, USA
      name:The University of Texas Graduate School of Biomedical Sciences, Houston, USA
      name:Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
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