Determined the copy number of gene fusions, and confirmed their chromosomal location by fluorescence in situ hybridization (FISH) (not shown). For small deletions and duplications, we determined the copy number of the relevant region Title Loaded From File relative to flanking regions from array CGH segmentation to assess whether the segment bearing the deletion or Title Loaded From File duplication had itself been duplicated. Earlier deletions and duplication showed a copy number shift of two or more whereas later events had a copy number shift of only one. Seven of the twelve fusion transcripts were classified as before endoreduplication; two, CTCF-SCUBE2 and BC041478-EXOSC10 were classified later. AGPAT5-MCPH1 and SUSD1-ROD1/PTBP3 and KLK5-CDH23 were undetermined, as their allelic copy number could not be resolved by array CGH or FISH. We were able to place seven deletions earlier, and these were all the homozygous deletions. Five deletions, all heterozygous, were placed later, with one undetermined. We could unambiguouslyTiming of Mutations in a Breast Cancer Genomeplace 14 small duplications relative to endoreduplication: seven earlier and seven later. To assign point mutations to one or two copies of particular chromosomes, we isolated the individual chromosomes in a cell sorter and re-sequenced the mutated exons (Fig. 4). We confirmed this analysis for selected 16985061 genes by measuring the relative number of mutant and wild-type copies using pyrosequencing (Fig. S2 in File S1). We were able to place 75 of the 85 previously described sequence-level mutations before or after endoreduplication, with only 10 undetermined. Of these ten, two were on a chromosome that was too small to be resolved in flow sorting, and 8 were not possible to score, either because they were found on single-copy genome segments, or they were found in a region where parent of origin could not be determined. Two reported mutations, in ZNF674 and HUWE1, were not found in our sample, therefore presumably occurred in other stocks of the line. They could therefore be classified as later (Fig. 3, Table 1 and Table S6 in File S2).Earlier and Later MutationsOverall, the proportion of mutations classified as occurring before endoreduplication 23148522 (earlier) was fairly similar for structural and point mutations (Table 1): 27/48 (56 ) of structural changes (translocations, deletions and duplications) and 34/75 (45 ) sequence-level changes were classed as earlier (Fig. 3 and Tables S4 7 in File S2). Among the structural mutations that could be classified, 13/22 (59 ) of chromosome translocations were in the earlier group, while 7/14 (50 ) of small duplications were earlier and 7/12 (58 ) of small deletions were earlier. For fusion genes, 7/9 (78 ) were classified earlier and, interestingly, all three in-frame fusion transcripts that could be classified were classified as earlier. Of the classifiable sequence-level mutations, 58 were missense mutations, of which 23/58 (40 ) fell early. To try to uncover `driver’ mutations within this group, we applied the Sorting Intolerant from Tolerant (SIFT) algorithm [23] to all of the point mutations, 47 of which could be scored by this method. Of the missense mutations predicted to be non-functional (tolerated) and so more likely to be random, 9/28 (32 ) were earlier, while 7/19 (37 ) mutations predicted to be functional (deleterious) were earlier (Table 1, Table S6 in File S2). Wood et al. [3] also identified genes likely to be drivers as `candidate cancer genes’ (CAN) based on their m.Determined the copy number of gene fusions, and confirmed their chromosomal location by fluorescence in situ hybridization (FISH) (not shown). For small deletions and duplications, we determined the copy number of the relevant region relative to flanking regions from array CGH segmentation to assess whether the segment bearing the deletion or duplication had itself been duplicated. Earlier deletions and duplication showed a copy number shift of two or more whereas later events had a copy number shift of only one. Seven of the twelve fusion transcripts were classified as before endoreduplication; two, CTCF-SCUBE2 and BC041478-EXOSC10 were classified later. AGPAT5-MCPH1 and SUSD1-ROD1/PTBP3 and KLK5-CDH23 were undetermined, as their allelic copy number could not be resolved by array CGH or FISH. We were able to place seven deletions earlier, and these were all the homozygous deletions. Five deletions, all heterozygous, were placed later, with one undetermined. We could unambiguouslyTiming of Mutations in a Breast Cancer Genomeplace 14 small duplications relative to endoreduplication: seven earlier and seven later. To assign point mutations to one or two copies of particular chromosomes, we isolated the individual chromosomes in a cell sorter and re-sequenced the mutated exons (Fig. 4). We confirmed this analysis for selected 16985061 genes by measuring the relative number of mutant and wild-type copies using pyrosequencing (Fig. S2 in File S1). We were able to place 75 of the 85 previously described sequence-level mutations before or after endoreduplication, with only 10 undetermined. Of these ten, two were on a chromosome that was too small to be resolved in flow sorting, and 8 were not possible to score, either because they were found on single-copy genome segments, or they were found in a region where parent of origin could not be determined. Two reported mutations, in ZNF674 and HUWE1, were not found in our sample, therefore presumably occurred in other stocks of the line. They could therefore be classified as later (Fig. 3, Table 1 and Table S6 in File S2).Earlier and Later MutationsOverall, the proportion of mutations classified as occurring before endoreduplication 23148522 (earlier) was fairly similar for structural and point mutations (Table 1): 27/48 (56 ) of structural changes (translocations, deletions and duplications) and 34/75 (45 ) sequence-level changes were classed as earlier (Fig. 3 and Tables S4 7 in File S2). Among the structural mutations that could be classified, 13/22 (59 ) of chromosome translocations were in the earlier group, while 7/14 (50 ) of small duplications were earlier and 7/12 (58 ) of small deletions were earlier. For fusion genes, 7/9 (78 ) were classified earlier and, interestingly, all three in-frame fusion transcripts that could be classified were classified as earlier. Of the classifiable sequence-level mutations, 58 were missense mutations, of which 23/58 (40 ) fell early. To try to uncover `driver’ mutations within this group, we applied the Sorting Intolerant from Tolerant (SIFT) algorithm [23] to all of the point mutations, 47 of which could be scored by this method. Of the missense mutations predicted to be non-functional (tolerated) and so more likely to be random, 9/28 (32 ) were earlier, while 7/19 (37 ) mutations predicted to be functional (deleterious) were earlier (Table 1, Table S6 in File S2). Wood et al. [3] also identified genes likely to be drivers as `candidate cancer genes’ (CAN) based on their m.