For whole genome sequencing, we had greater than 60�� average coverage for the primary cancer whole genome sample and 30�� for the metastatic genome. The whole genome sequencing was used for identifying larger scale genetic aberrations such as copy number variation, allelic imbalances, rearrangements, and other classes of structural rearrangements. After alignment, we conducted variant calling to identify somatic muta tions and other classes of genetic aberrations. This in cluded somatic mutations, insertion deletions, CNVs, loss of heterozygosity regions, and cancer rearrangements. As a control for single nucleotide variant calling, we geno typed the samples with Affymetrix 6. 0 single nucleotide polymorphism arrays. we compared the genotypes to the identified SNPS from the sequence data.

The con cordance of exome and whole genome SNP data to the array data was 99%. Coding region mutations and validation with deep sequencing We identified mutations that occurred in exons and in tronic mutations within 100 bases of the exon boundary and the results are summarized in Additional file 1 Table S2. As noted previously, the tumor samples had complex composition that reduced the sequence cover age of some mutations. We proceeded with an additional round of targeted sequencing to validate these mutations and determine their presence in both tumors. We designed an assay for deep targeted resequencing that covered approximately 300 bases around the specific mutation loci. The average targeted sequencing coverage for each putative mutation or loci was 278�� for the normal, 251�� for the primary tumor and 152�� for the metastasis.

Between the two tumors, we independently validated a total of 77 mutations that occurred within or proximal to exons. Vali dated genetic aberrations included non synonymous mutations, synonymous mutations, insertions, or deletions. With the targeted sequencing data, we de termined the mutation allelic frequency between the primary tumor and metastasis for each mutation. This involves determining the fraction of a sequence read with a mutation in comparison to the reference se quence Anacetrapib reads. We were able to identify which mutations were common or exclusive to the primary tumor versus the metastasis. Among the 77 validated mutations, the distribution was such that mutations were generally unique either to the primary tumor or metastatic site.

For example, the primary tumor had eight mutations that were not present in the metastasis while the metas tasis had 37 mutations not present in the primary tumor. Common to both cancers were 32 mutations. Given the interval of three years prior to the detection of the metastasis, there is a possibility that the metastasis specific mutations occurred independently from the pri mary tumor. Mutations specific to the primary tumor that were not present in the ovarian metastasis may have been the result of random genetic drift.