Establishment of Genomic/Epigenomic Analysis Methods Based on Next Generation Sequencing, and Genomic Alterations Discovery in Korean Triple Negative Breast Cancer

Abstract

The development of Next Generation Sequencing (NGS) technology makes it possible to analyze huge amount of sequencing data with low cost, and stimulates worldwide genetic research field. Therefore, NGS will be applicable to diagnose or treat the patient with genetic disease (e.g. cancer) in the near future. NGS can be used in various genome-wide genomic/epigenomic analyzes including Targeted exome sequencing for analysing only exon regions of interesting genes, RNA sequencing for analysing RNA expression profiles and splicing variants, Bisulfite sequencing for analysing DNA methylation and Chromatin Immunoprecipitation sequencing (ChIP-seq) for analysing histone modification. While the cost for sequencing data generation rapidly decreased with NGS technology progression, the cost for data storage and data analysis increases proportionally to the amount of sequencing data. Hence, it is important to perform NGS within interesting regions depending on the purpose of each research, increasing the efficiency of time and cost. In the first part of this study, the efficiency of a methylated DNA immunoprecipitation bisulfite sequencing (MeDIP-BS) method, that involves a combination of methylated region enrichment using 5-mC antibodies and bisulfite conversion, was evaluated. By taking the advantage of the low cost of Methylated DNA Immunoprecipitation sequencing (MeDIP-seq) and the high resolution of Whole Genome Bisulfite sequencing (WG-BS), this method not only remarkably improves cost effectiveness, but also dramatically enhances analysis resolution, achieving base-pair resolution. In addition, by comparing this method to WG-BS, MeDIP-Seq, and Targeted Bisulfite sequencing (Targeted-BS) in analyzes using human liver and stomach samples, it is proved that MeDIP-BS is applicable for clinical diagnostics, guaranteeing cost-effective high read depth and high-resolution genome-wide DNA methylation analysis. In the second part of this study, genomic alterations of Korean triple negative breast cancer (TNBC) patients were profiled by targeted exome sequencing which aims at analysing target exome regions. This method had revolutionized human clinical cancer diagnosis, cancer-causing mechanism studies and processes for identifying therapeutic targets due to its cost-effectiveness compared with whole genome or whole exome NGS. The targeted exome sequencing is very advantageous at providing more reliable accuracy of mutation and copy number alteration analysis by generating sufficiently deeper coverage of sequencing reads in target exon regions at a relatively lower cost compared with whole exome NGS. In particular, HaloPlex target enrichment system had already been substantiated for its advantageous usefulness in the targeted exome NGS due to its high efficiency upon capturing the targeted regions on the exome. Through this study, for the first time to our knowledge, it is revealed that mutation and copy number variation landscapes on targeted regions for 368 cancer-associated genes from 70 Korean TNBC patients. Futhermore, some homozygous deletion genes have significant correlation with prognosis, suggesting the potential role as prognostic biomarker.