Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of the SKBR3 breast cancer cell line

Maria Nattestad, Sara Goodwin, Karen Ng, Timour Baslan, Fritz J. Sedlazeck, Philipp Rescheneder, Tyler Garvin, Han Fang, James Gurtowski, Elizabeth Hutton, Elizabeth Tseng, Chen-Shan Chin, Timothy Beck, Yogi Sundaravadanam, Melissa Kramer, Eric Antoniou, John D. McPherson, James Hicks, William Richard McCombie, Michael C. Schatz

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences, and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available with nearly 20,000 variants present, most of which were missed by prior efforts. Surrounding the important HER2 locus, we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the transcriptome and sheds new light on the complexity of cancer progression.

Analysis Manuscripts
» Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line
Nattestad et al. (2017) bioRxiv. doi:
» Accurate detection of complex structural variations using single molecule sequencing
Sedlazeck, Rescheneder et al (2017) bioRxiv. doi:
» SplitThreader: Exploration and analysis of rearrangements in cancer genomes
Nattestad et al (2016) bioRxiv. doi:
» Ribbon: Visualizing complex genome alignments and structural variation
Nattestad et al (2016) bioRxiv. doi:

Raw Reads
» Raw PacBio and Illumina reads are available in the NCBI SRA under under BioProject PRJNA476239

PacBio-based Analysis
» Reads aligned with NGMLR:
» Variants discovered with Sniffles:
» SplitThreader analysis of Copy number variants + long range Sniffles variants:
» Ribbon display of SKBR3 structural variations:
» FALCON assembly of PacBio reads:
» Assemblytics analysis of FALCON assembly:

Illumina-based Analysis
» Illumina PE550 reads aligned with BWA-MEM:
» Delly SVs:
» Lumpy SVs:
» Manta SVs:
» PopIns SVs:
» SURVIVOR consensus SVs:
» Allpaths-LG assembly of the Illumina reads:
» Assemblytics analysis of ALLPATHS-LG assembly: