title: Amplification and thrifty single-molecule sequencing of recurrent somatic structural variations.
abstract: Deletion of tumor-suppressor genes as well as other genomic rearrangements pervade cancer genomes across numerous types of solid tumor and hematologic malignancies. However, even for a specific rearrangement, the breakpoints may vary between individuals, such as the recurrent CDKN2A deletion. Characterizing the exact breakpoints for structural variants (SVs) is useful for designating patient-specific tumor biomarkers. We propose AmBre (Amplification of Breakpoints), a method to target SV breakpoints occurring in samples composed of heterogeneous tumor and germline DNA. Additionally, AmBre validates SVs called by whole-exome/genome sequencing and hybridization arrays. AmBre involves a PCR-based approach to amplify the DNA segment containing an SV's breakpoint and then confirms breakpoints using sequencing by Pacific Biosciences RS. To amplify breakpoints with PCR, primers tiling specified target regions are carefully selected with a simulated annealing algorithm to minimize off-target amplification and maximize efficiency at capturing all possible breakpoints within the target regions. To confirm correct amplification and obtain breakpoints, PCR amplicons are combined without barcoding and simultaneously long-read sequenced using a single SMRT cell. Our algorithm efficiently separates reads based on breakpoints. Each read group supporting the same breakpoint corresponds with an amplicon and a consensus amplicon sequence is called. AmBre was used to discover CDKN2A deletion breakpoints in cancer cell lines: A549, CEM, Detroit562, MOLT4, MCF7, and T98G. Also, we successfully assayed RUNX1-RUNX1T1 reciprocal translocations by finding both breakpoints in the Kasumi-1 cell line. AmBre successfully targets SVs where DNA harboring the breakpoints are present in 1:1000 mixtures.
authors.affiliation: Bioinformatics and Systems Biology Program, University of California San Diego, La Jolla, California 92093, USA;
authors.forename: Anand
authors.initials: A
authors.lastname: Patel
authors.forename: Richard
authors.initials: R
authors.lastname: Schwab
authors.forename: Yu-Tsueng
authors.initials: YT
authors.lastname: Liu
authors.forename: Vineet
authors.initials: V
authors.lastname: Bafna
mesh_terms: D000465:Algorithms; D002869:Chromosome Aberrations; D056905:Chromosome Breakpoints; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D059472:Exome / Q000235:genetics; D015894:Genome, Human; D056914:Genomic Structural Variation / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D061986:MCF-7 Cells; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D014178:Translocation, Genetic
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16