Abstract 3603: Megabase-scale determination of complex genetic aberrations of primary cancer genomes at individual DNA molecule resolution
Authors: Lau Billy , Bell John , Greer Stephanie , Zheng Grace , Wood Christina , Ji Hanlee 
Published: July 2016

Cancer is a disease of the genome caused by genetic aberrations such as point mutations and rearrangements. To identify these genetic aberrations, cancer studies rely on DNA sequencing of short reads from highly fragmented DNA molecules that are typically less than 500 bases long. However, genomic structural alterations and context of genetic aberrations are much larger and cannot be effectively analyzed. To characterize such alterations with completeness, the contiguous relationship of variants from a given chromosome homologue must be determined; this analysis process is referred to as genome phasing. In this study, we demonstrate the significant advantages of genome phasing; we utilize a new technology called linked-read sequencing that enables tracking of individual DNA sequence reads to the input high molecular weight DNA at single-molecule resolution through the use of barcoded reads. We then reconstruct at megabase-scale the complex genomic somatic events derived from the original paternal or maternal chromosome homologues and demonstrate new insight into the undiscovered details of cancer genome alterations. We apply this analysis to primary gastrointestinal cancers from surgical resections; our study is a unique achievement given that this type of phased genome sequencing analysis has only been previously conducted on cancer cell lines.

For this study, we focus on specific categories of megabase-scale genetic aberrations: (1) copy number variants (CNV) derived from segmental duplications, (2) structural rearrangements, and (3) aneuploidy. As an example from gastric cancer metastases, we identified a set of unique and site-specific FGFR2 amplification events corresponding to segmental duplications. The structural details of this amplification were only evident using barcoded sequence reads and phased analysis. Secondly, in a primary colorectal adenocarcinoma we resolved a complex recombination event that covered the majority of the chromosome 5 q-arm. It contained a set of complex rearrangements that are difficult to determine by short read sequencing but can be identified using linked reads. This rearrangement event arose from a combination of a 80Mb homologous recombination that contained a small interval ∼2.6Mb deletion. The allelic loss of two candidate cancer drivers, SMAD5 and TGFB, occurred in the affected interval. Finally, to assess aneuploidy we relied on the imbalanced ratios between paternal and maternal chromosomal homologues to generate contiguous blocks of variants on the order of tens and up to hundreds of megabases long. We performed this type of analysis on a number of cancer genomes derived from primary colorectal adenocarcinomas, and were able to robustly determine the phased single nucleotide variant content and large-scale genomic alterations involving entire chromosome arms.

Citation Format: Billy Lau, John Bell, Stephanie Greer, Grace Zheng, Christina Wood, Hanlee Ji. Megabase-scale determination of complex genetic aberrations of primary cancer genomes at individual DNA molecule resolution. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3603.