G CTNNA1 and NFKBIA (both earlier, see above). Others have cancer-relevant functions, such as steroid hormone synthesis (HSD17B8, earlier), and covalent modification of histones (HUWE1, IPO7, MLL4, PAXIP1, PRKAA2, all later except PAXIP1) (Table S7 in File S2).Applicability to Sequencing DataOur theoretical framework and statistical methods could be applied, in a modified form, to sequencing data from other endoreduplicated cell lines and primary tumours, indeed the idea of placing mutations before or after a duplication event has already been exploited [1,18]. Endoreduplication is a common process in epithelial cancers, estimated to occur in more than 50 of breast cancers [17,18]. Endoreduplicated genomes can often be identifed by copy number and allele ratios [18], for example, a large proportion of a recently-endoreduplicated genome will often be present either in four copies and heterozygous, or two homozygous copies (Fig. 4). We relied on flow sorting of chromosomes to quantify our mutations, but the proportion of mutant and reference alleles could be deduced, for example, by counting reads from deep massively-parallel sequencing. Earlier mutations will usually be homozygous in diploid regions, or account for approximately 50 of mutant reads in tetraploid regions. Distinguishing between earlier and later events in 16985061 large datasets may help identify genes or pathways that must be mutated earlier or later in a given tumour type.For sequencing, exons with flanking intronic sequence were amplified using published primer sequences [3]. Reactions were performed as above using 25 ng SPDP biological activity flow-sorted and amplified chromosomes or HCC1187 whole genomic DNA as a target. PCR products were cleaned up using Nucleofast 96 PCR cleanup kit (Clontech, Mountain View, CA) and sequenced in both directions using the same primers as for amplification with BigDye v3.1 (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions on an ABI 3700 capillary DNA sequencer. SNP6 data [20] are available online (www.sanger.ac.uk/cgibin/genetics/CGP). Data were viewed as PICNIC-segmented graphical output [43].Supporting InformationFile S1 Figures S1 and S2. Figures S1 and S2 23148522 are provided in a Sudan I price single pdf document. Figure S1. Segmentation by PICNIC algorithm reveals `Parent A’ and `Parent B’ origin of segments of chromosome 13. Figure S2. Pyrosequencing confirmation of the HSD17B8 mutation. (PDF) File S2 Tables S1 7. Tables provided as a single spreadsheet in Excel format. Table S1, cytogenetic descriptions of genome rearrangements in HCC1187, from ref. 12. Table S2, array-CGH data segmented PICNIC algorithm. Table S3, genome segments originally identified by array painting in ref. 12, with breakpoints refined by comparison with array CGH data in table S2. Table S4, Expressed Fusion Genes. Table S5, Deletions and duplications of less than 2 Mb, identified from array CGH. Table S6, Sequencelevel mutations, with comments and annotations as described in the text. Table S7, all genes affected by mutation, with timing, recurrence of mutation in breast cancer, and brief gene annotation. (XLS) File SConclusionIn conclusion, we provide evidence that, in this cell line, chromosome instability and rearrangement was not a late and irrelevant event, and that the great majority of inactivating mutations and expressed gene fusions appear to have happened early, and this suggests that most of them were selected.Details of statistical model.(PDF)Materials and MethodsCel.G CTNNA1 and NFKBIA (both earlier, see above). Others have cancer-relevant functions, such as steroid hormone synthesis (HSD17B8, earlier), and covalent modification of histones (HUWE1, IPO7, MLL4, PAXIP1, PRKAA2, all later except PAXIP1) (Table S7 in File S2).Applicability to Sequencing DataOur theoretical framework and statistical methods could be applied, in a modified form, to sequencing data from other endoreduplicated cell lines and primary tumours, indeed the idea of placing mutations before or after a duplication event has already been exploited [1,18]. Endoreduplication is a common process in epithelial cancers, estimated to occur in more than 50 of breast cancers [17,18]. Endoreduplicated genomes can often be identifed by copy number and allele ratios [18], for example, a large proportion of a recently-endoreduplicated genome will often be present either in four copies and heterozygous, or two homozygous copies (Fig. 4). We relied on flow sorting of chromosomes to quantify our mutations, but the proportion of mutant and reference alleles could be deduced, for example, by counting reads from deep massively-parallel sequencing. Earlier mutations will usually be homozygous in diploid regions, or account for approximately 50 of mutant reads in tetraploid regions. Distinguishing between earlier and later events in 16985061 large datasets may help identify genes or pathways that must be mutated earlier or later in a given tumour type.For sequencing, exons with flanking intronic sequence were amplified using published primer sequences [3]. Reactions were performed as above using 25 ng flow-sorted and amplified chromosomes or HCC1187 whole genomic DNA as a target. PCR products were cleaned up using Nucleofast 96 PCR cleanup kit (Clontech, Mountain View, CA) and sequenced in both directions using the same primers as for amplification with BigDye v3.1 (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions on an ABI 3700 capillary DNA sequencer. SNP6 data [20] are available online (www.sanger.ac.uk/cgibin/genetics/CGP). Data were viewed as PICNIC-segmented graphical output [43].Supporting InformationFile S1 Figures S1 and S2. Figures S1 and S2 23148522 are provided in a single pdf document. Figure S1. Segmentation by PICNIC algorithm reveals `Parent A’ and `Parent B’ origin of segments of chromosome 13. Figure S2. Pyrosequencing confirmation of the HSD17B8 mutation. (PDF) File S2 Tables S1 7. Tables provided as a single spreadsheet in Excel format. Table S1, cytogenetic descriptions of genome rearrangements in HCC1187, from ref. 12. Table S2, array-CGH data segmented PICNIC algorithm. Table S3, genome segments originally identified by array painting in ref. 12, with breakpoints refined by comparison with array CGH data in table S2. Table S4, Expressed Fusion Genes. Table S5, Deletions and duplications of less than 2 Mb, identified from array CGH. Table S6, Sequencelevel mutations, with comments and annotations as described in the text. Table S7, all genes affected by mutation, with timing, recurrence of mutation in breast cancer, and brief gene annotation. (XLS) File SConclusionIn conclusion, we provide evidence that, in this cell line, chromosome instability and rearrangement was not a late and irrelevant event, and that the great majority of inactivating mutations and expressed gene fusions appear to have happened early, and this suggests that most of them were selected.Details of statistical model.(PDF)Materials and MethodsCel.
