Abstract 850: Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using single molecule sequencing
Authors: Nattestad Maria , Ng Karen , Goodwin Sara , Baslan Timour , Sedlazeck Fritz , Gurtowski James , Hutton Elizabeth , Sundaravadanam Yogi , Garvin Tyler , Alford Marley , Tseng Elizabeth , Rescheneder Philipp , Chin Jason , Beck Timothy , Kramer Melissa , McPherson John , Hicks James , Schatz Michael C. , McCombie William R. 
Published: July 2016

Genomic instability is one of the hallmarks of cancer, leading to widespread copy number variations, chromosomal fusions, and other structural variations in many cancers. The breast cancer cell line SK-BR-3 is an important model for HER2+ breast cancers, which are among the most aggressive forms of the disease and affect one in five cases. Through short read sequencing, copy number arrays, and other technologies, the genome of SK-BR-3 is known to be highly rearranged with many copy number variations, including an approximately twenty-fold amplification of the HER2 oncogene, along with numerous other amplifications and deletions. However, these technologies cannot precisely characterize the nature and context of the identified genomic events and other important mutations may be missed altogether because of repeats, multi-mapping reads, and the failure to reliably anchor alignments to both sides of a variation.

To address these challenges, we have sequenced SK-BR-3 using PacBio long read technology. Using the new P6-C4 chemistry, we generated more than 70X coverage of the genome with average read lengths of 9-13kb (max: 71kb). Using Lumpy for split-read alignment analysis, as well as our novel assembly-based algorithms for finding complex variants, we have developed a detailed map of structural variations in this cell line. Taking advantage of the newly identified breakpoints and combining these with copy number assignments, we have developed an algorithm to reconstruct the mutational history of this cancer genome. From this we have characterized the amplifications of the HER2 region, discovering a complex series of nested duplications and translocations between chr17 and chr8, two of the most frequent translocation partners in primary breast cancers. We have also carried out full-length transcriptome sequencing using PacBio's Iso-Seq technology, which has revealed a number of previously unrecognized gene fusions and isoforms. Combining long-read genome and transcriptome sequencing technologies enables an in-depth analysis of how changes in the genome affect the transcriptome, including how gene fusions are created across multiple chromosomes. This analysis has established the most complete cancer reference genome available to date, and is already opening the door to applying long-read sequencing to patient samples with complex genome structures.

Citation Format: Maria Nattestad, Karen Ng, Sara Goodwin, Timour Baslan, Fritz Sedlazeck, James Gurtowski, Elizabeth Hutton, Yogi Sundaravadanam, Tyler Garvin, Marley Alford, Elizabeth Tseng, Philipp Rescheneder, Jason Chin, Timothy Beck, Melissa Kramer, John McPherson, James Hicks, Michael C. Schatz, William R. McCombie. Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using single molecule sequencing. [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 850.