Simultaneous estimation of detection sensitivity and absolute copy number from digital PCR serial dilution |
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| Institution: | 1. Blood Systems Research Institute, San Francisco, CA 94118, USA;2. Department of Laboratory Medicine, University of California at San Francisco, San Francisco, CA 94107, USA;1. Laboratório de Nanossensores, Instituto de Pesquisa & Desenvolvimento, Universidade do Vale do Paraíba, São José dos Campos, São Paulo, Brazil;2. Laboratório de Biologia Celular e Molecular de Fungos, Instituto de Pesquisa & Desenvolvimento, Universidade do Vale do Paraíba, São José dos Campos, São Paulo, Brazil;3. Laboratório de Bionanotecnologia, Instituto de Pesquisa & Desenvolvimento, Universidade do Vale do Paraíba, São José dos Campos, São Paulo, Brazil;1. Division of Hematology and Clinical Immunology and Bone Marrow Transplant Program, Department of Medicine;2. Division of Microbiology, Department of Experimental Medicine, University of Perugia, Perugia, Italy;;3. Blood Bank, Ospedale Santa Maria della Misericordia, Perugia, Italy;;4. Immunotherapy Laboratory, Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland; and;5. Division of Hematology and Bone Marrow Transplant Unit, Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy;1. School of Pharmacy, Iwaki Meisei University, Iino, Chuodai, Iwaki, 970-8551, Fukushima, Japan;2. School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, 192-0392, Tokyo, Japan;3. Graduate School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku, 108-8641, Tokyo, Japan;1. Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University of School of Medicine, Seoul, Republic of Korea;2. Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University of School of Medicine, Seoul, Republic of Korea |
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| Abstract: | Digital polymerase chain reaction (dPCR) is a refinement of the conventional PCR approach to nucleic acid detection and absolute quantification. Digital PCR works by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions. Current quantification methods rely on the assumption that the PCR reactions are always able to detect single target molecules. When the assumption does not hold, the copy numbers will be severely underestimated. We developed a novel dPCR quantification method which determines whether the single copy assumption is violated or not by simultaneously estimating the assay sensitivity and the copy numbers using serial dilution data sets. The implemented method is available as an R package “digitalPCR”. |
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