title: A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens.
abstract: The ability of droplet digital PCR (ddPCR) to accurately determine the concentrations of amplifiable targets makes it a promising platform for measuring copy number alterations (CNAs) in genomic biomarkers. However, its application to clinical samples, particularly formalin-fixed paraffin-embedded specimens, will require strategies to reliably determine CNAs in DNA of limited quantity and quality. When applied to cancerous tissue, those methods must also account for global genetic instability and the associated probability that the abundance(s) of one or more chosen reference loci do not represent the average ploidy of cells comprising the specimen. Here we present an experimental design strategy and associated data analysis tool that enables accurate determination of CNAs in a panel of biomarkers using multiplexed ddPCR. The method includes strategies to optimize primer and probes design to cleanly segregate droplets in the data output from reaction wells amplifying multiple independent templates, and to correct for bias from artifacts such as DNA fragmentation. We demonstrate how a panel of reference loci can be used to determine a stable CNA-neutral benchmark. These innovations, when taken together, provide a comprehensive strategy that can be used to reliably detect biomarker CNAs in DNA extracted from either frozen or FFPE tissue biopsies.
authors.affiliation: Department of Oral Medical Biological Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.|Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada.
authors.forename: Curtis B
authors.initials: CB
authors.lastname: Hughesman
authors.affiliation: Department of Oral Medical Biological Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: X J David
authors.initials: XJ
authors.lastname: Lu
authors.affiliation: Department of Oral Medical Biological Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: Kelly Y P
authors.initials: KY
authors.lastname: Liu
authors.affiliation: Department of Oral Medical Biological Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Oral Medical Biological Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, V6T 2B5, Canada.|Department of Integrative Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, V5Z 1L3, Canada.
authors.forename: Catherine F
authors.initials: CF
authors.lastname: Poh
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada.|RES'EAU NSERC Research Network, Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: Charles
authors.initials: C
authors.lastname: Haynes
mesh_terms: D001706:Biopsy; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D056915:DNA Copy Number Variations / Q000235:genetics; D053938:DNA Fragmentation; D018628:Gene Dosage / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D011003:Ploidies
chemical_list: D005819:Genetic Markers; D004247:DNA