Amplicon Sequencing for Genomic Analysis
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Corresponding Organization : University of Washington
Other organizations : Seattle Children's Hospital, Meyer Children's Hospital, University of Florence, Stanford University, Manipal Academy of Higher Education, Kasturba Medical College, Manipal, Children's Hospital of Eastern Ontario, University Children's Hospital Tübingen, Sinai Hospital, University of Toronto, University Hospitals Case Medical Center, Center for Human Genetics, Saint Louis University, University of Vermont, Royal North Shore Hospital, Belfast Health and Social Care Trust, Children's Hospital of Los Angeles, University of Southern California, Tel Aviv University, Schneider Children's Medical Center, Griffith University, University of Michigan–Ann Arbor, Pediatrics and Genetics, KK Women's and Children's Hospital, University of British Columbia, University Hospital of Lausanne, Greenwood Genetic Center, Garrahan Hospital, Children's Hospital of Michigan, Wayne State University, KU Leuven, St George’s University Hospitals NHS Foundation Trust, Institute of Cancer Research, University of Minnesota, University of Florida, Liverpool Hospital, Providence Sacred Heart Medical Center, Academic Medical Center, University of Amsterdam, Pontificia Universidad Católica de Chile, Hospital Luis Calvo Mackenna, Alfred I. duPont Hospital for Children, University of Rochester, Santa Maria Nuova Hospital, University of California, San Francisco, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Koç University, Howard Hughes Medical Institute, Cedars-Sinai Medical Center, Harbor–UCLA Medical Center
Variable analysis
- Primer sequences used for locus-specific amplification of genomic DNA
- Amplicon size (ranging from 290 to 310 bp)
- Amplicon sequence
- Previously published methods for amplicon sequencing (reference 48)
- Use of Primer3Plus software for primer design
- Removal of small DNA fragments using Agencourt AMPure XP
- Quantification of amplicons using Quant-iT PicoGreen dsDNA reagent
- Pooling of amplicons at equimolar ratios
- Emulsion PCR using GS Junior Titanium emPCR kit (Lib-A kit)
- Pyrosequencing on a GS Junior sequencer
- Data analysis using GS Amplicon Variant Analyzer version 3.0 software
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