Dataset Information

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.

SUBMITTER: Hampton OA

PROVIDER: S-EPMC3185296 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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

Hampton Oliver A OA Koriabine Maxim M Miller Christopher A CA Coarfa Cristian C Li Jian J Den Hollander Petra P Schoenherr Caroline C Carbone Lucia L Nefedov Mikhail M Ten Hallers Boudewijn F H BF Lee Adrian V AV De Jong Pieter J PJ Milosavljevic Aleksandar A

Cancer genetics 20110801 8

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 ...[more]

PMID: 21962895

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Project description:BACKGROUND:We recently described Hi-Plex, a highly multiplexed PCR-based target-enrichment system for massively parallel sequencing (MPS), which allows the uniform definition of library size so that subsequent paired-end sequencing can achieve complete overlap of read pairs. Variant calling from Hi-Plex-derived datasets can thus rely on the identification of variants appearing in both reads of read-pairs, permitting stringent filtering of sequencing chemistry-induced errors. These principles underly ROVER software (derived from Read Overlap PCR-MPS variant caller), which we have recently used to report the screening for genetic mutations in the breast cancer predisposition gene PALB2. Here, we describe the algorithms underlying ROVER and its usage. RESULTS:ROVER enables users to quickly and accurately identify genetic variants from PCR-targeted, overlapping paired-end MPS datasets. The open-source availability of the software and threshold tailorability enables broad access for a range of PCR-MPS users. METHODS:ROVER is implemented in Python and runs on all popular POSIX-like operating systems (Linux, OS X). The software accepts a tab-delimited text file listing the coordinates of the target-specific primers used for targeted enrichment based on a specified genome-build. It also accepts aligned sequence files resulting from mapping to the same genome-build. ROVER identifies the amplicon a given read-pair represents and removes the primer sequences by using the mapping co-ordinates and primer co-ordinates. It considers overlapping read-pairs with respect to primer-intervening sequence. Only when a variant is observed in both reads of a read-pair does the signal contribute to a tally of read-pairs containing or not containing the variant. A user-defined threshold informs the minimum number of, and proportion of, read-pairs a variant must be observed in for a 'call' to be made. ROVER also reports the depth of coverage across amplicons to facilitate the identification of any regions that may require further screening. CONCLUSIONS:ROVER can facilitate rapid and accurate genetic variant calling for a broad range of PCR-MPS users.

Dataset Information

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

Publications

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

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