DNA methylation profile of triple negative breast cancer-specific genes (MBD-Seq). Homo sapiens

This part of the study was used to validate our findings comparing the DNA methylation in lymph node metastasis to that in normal tissue. We compared our findings (comparing LN versus NAT) with MBDcap sequencing data provided by Dr. Clare Stirzaker and Prof. Susan Clark from the Garvan Institute Sydney. This analysis provides regional DNA methylation information therefore a strict validation with our CpG-specific analysis was not possible. The regions that were analysed using the MBDcap data included our genes of interest (83 genes that are associated with LN metastasis, identified in 16) starting 2kb upstream of the first transcription start site (TSS) to the 3’ end of the genes. These regions are broken up into 1kb tiles. Methylation differences between LN and NAT samples were assessed using a Student’s t-test (using the R base package) (p<0.05). Overall design: Genome wide profiling of 7 Lymph node metastatic triple negative breast tumours was performed using MBDCap-seq. Methylation in these samples was compared to normal breast samples that can be found at GSE58020 (GSM1399530,GSM1399522,GSM1399520,GSM1399524). To account for inter patient and inter-region variation, read counts were normalised to total library size and then divided by read counts in a 100% methylated control sample (see GSE58020 sample GSM1399545).