SKBR3 PacBio Sequencing and Assembly

Cold Spring Harbor Laboratory and Ontario Institute for Cancer Research

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 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). PacBio read coverage is highly correlated with the copy number assignments made using short read sequencing technologies, although the long reads provide more consistent coverage across repetitive elements. Furthermore, using the structural variation analysis program LUMPY and our new hybrid mapping and de novo assembly algorithm for analyzing split-read alignments, we have developed a detailed map of structural variations in this cell line. We have tentatively identified more than 900 intra-chromosomal and 300 inter-chromosomal variations, including many of the previously known gene fusions in SK-BR-3. Taking advantage of the newly identified breakpoints, 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. To our knowledge, this establishes the most complete cancer reference genome to date.

See the slides from the PacBio Workshop at AGBT

Data Usage Agreement

Users of these for genome wide analysis prior to our publication must agree to co-authorship as specified by the Toronto agreement.

PacBio Read Length Distribution

View alignments in bam.iobio.io

By clicking these links, you agree to the Toronto agreement:
skbr3.pacbio.fastq.gz (196GB)
SKBR3_Feb17_GRCh38.sorted.bam (280GB)
FALCON assembly can be downloaded from DNAnexus