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

Graduate Program in Structural and Computational Biology and Molecular Biophysics, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
Cancer Genetics (Impact Factor: 2.98). 08/2011; 204(8):447-57. DOI: 10.1016/j.cancergen.2011.07.009
Source: PubMed


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.

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    • "Cloning-free methods for linking reads together over long stretches of DNA range from 2 to 20 kb with increasing need for input DNA for increasing insert sizes. Cloning-based libraries such as fosmid libraries can further increase the insert size17. By incorporating a known distance between two sequence reads during construction of the library, that information can be utilized when deciphering the structure of a known genome, ordering the contigs built from assembly, to span or aid mapping in low complexity regions. "
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    • "In ZR-75-30, using structural analysis, we found half of the six expressed fusions detected by Robinson DR et al. [15], while, using cDNA sequencing, they found three of the nine we detected—both figures suggest the true total might be around 18. This is consistent with recent, probably incomplete, figures from other cell lines: 20 expressed fusions have been verified in MCF7, with several more predicted computationally [6,13,15,40]; 43 have been found in BT474 and 13 in SKBR3 [13]. "
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    ABSTRACT: Background It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements. Results We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing—55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads). From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer—in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator. Conclusions This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports the view that gene breakage and gene fusion are important classes of mutation in breast cancer, with a typical breast cancer expressing many fusion genes.
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    • "In this respect, Fosills are similar to Fosmid ''diTags'' (Hampton et al. 2011). The principal advantage of our method is that it allows much longer sequencing reads (up to 2 3 101 bases in the current study, but even longer reads are possible), whereas the EcoP15I digest strictly limits the diTags to 2 3 26 bases. "
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