Long-range massively parallel mate pair sequencing detects distinct mutations and similar patterns of structural mutability in two breast cancer cell lines

Abstract

Cancer genomes frequently undergo genomic instability resulting in accumulation of chromosomal rearrangement. To date, one of the main challenges has been to confidently and accurately identify these rearrangements by using short-read massively parallel sequencing. We were able to improve cancer rearrangement detection by combining two distinct massively parallel sequencing strategies: fosmid-sized (36 kb on average) and standard 5 kb mate pair libraries. We applied this combined strategy to map rearrangements in two breast cancer cell lines, MCF7 and HCC1954. We detected and validated a total of 91 somatic rearrangements in MCF7 and 25 in HCC1954, including genomic alterations corresponding to previously reported transcript aberrations in these two cell lines. Each of the genomes contains two types of breakpoints: clustered and dispersed. In both cell lines, the dispersed breakpoints show enrichment for low copy repeats, while the clustered breakpoints associate with high copy number amplifications. Comparing the two genomes, we observed highly similar structural mutational spectra affecting different sets of genes, pointing to similar histories of genomic instability against the background of very different gene network perturbations.

Figure 1

(A) Circular visualizations of the MCF7 and HCC1954 genomes obtained using Circos [52] software. Chromosomes are individually colored with centromeres in white. Copy number variation is plotted with gains in blue and losses in red. The colored rearrangements depict breast cancer-specific somatic mutations from the combined fosmid-sized and 5Kb mate pair libraries. Green lines denote intrachromosomal and purple lines denote interchromosomal rearrangements. (B) Venn diagrams comparing the numbers of fosmid-sized and 5Kb mate pair rearrangements, PCR primer designs, PCR assays that produced breakpoint amplicon versus no amplification product, and rearrangements that are validated as breast cancer-specific mutation versus normal structural variation in the MCF7 and HCC1954 genomes.

Figure 2

(A) Arc visualizations of the largest MCF7 and HCC1954 breakpoint cliques and their association with copy number amplification. Chromosome cytobands are shaded and labeled. The colored rearrangements depict breast tumor intrachromosomal (green) and interchromosomal (purple) mutations. Copy number counts are plotted with gains in blue, losses in red, and normal diploid in grey (count scales are from zero to MCF7: max = 60; HCC1954: max = 15). (B) MCF7 and HCC1954 log₂ copy number plots of Affymetrix 100k SNP chip arrays [34] (top) and Illumina mate pair mapped sequence counts (bottom); gains are plotted in blue, losses in red, and normal diploid in grey. Highlighted regions correspond to the largest breakpoint cliques from A.