Amplicon sequencing was performed using previously published methods (48 (link)). We performed locus-specific amplification of genomic DNA followed by GS Junior sequencing (Roche). We designed fusion primers containing genome-specific sequences along with distinct multiplex identifier sequences (used to differentiate samples being run together on the same plate) and sequencing adapters, to generate amplicons ranging in size from 290 to 310 bp, using Primer3Plus software. Primer sequences are provided in Supplemental Table 4. Small DNA fragments were removed with Agencourt AMPure XP (Beckman Coulter) according to the manufacturer’s protocol. All amplicons were quantified with the Quant-iT PicoGreen dsDNA reagent (Life Technologies), pooled at equimolar ratios, amplified by emulsion PCR using the GS Junior Titanium emPCR kit (Lib-A kit, Roche Applied Science), and pyrosequenced in the sense and antisense strands on a GS Junior sequencer following the manufacturers’ instructions. Data were analyzed using GS Amplicon Variant Analyzer version 3.0 software.
Mirzaa G., Timms A.E., Conti V., Boyle E.A., Girisha K.M., Martin B., Kircher M., Olds C., Juusola J., Collins S., Park K., Carter M., Glass I., Krägeloh-Mann I., Chitayat D., Parikh A.S., Bradshaw R., Torti E., Braddock S., Burke L., Ghedia S., Stephan M., Stewart F., Prasad C., Napier M., Saitta S., Straussberg R., Gabbett M., O’Connor B.C., Keegan C.E., Yin L.J., Lai A.H., Martin N., McKinnon M., Addor M.C., Boccuto L., Schwartz C.E., Lanoel A., Conway R.L., Devriendt K., Tatton-Brown K., Pierpont M.E., Painter M., Worgan L., Reggin J., Hennekam R., Tsuchiya K., Pritchard C.C., Aracena M., Gripp K.W., Cordisco M., Van Esch H., Garavelli L., Curry C., Goriely A., Kayserilli H., Shendure J., Graham J J.r., Guerrini R, & Dobyns W.B. (2016). PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution. JCI Insight, 1(9), e87623.
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
Primer sequences used for locus-specific amplification of genomic DNA
dependent variables
Amplicon sequences generated by GS Junior sequencing
control variables
Previously published methods for amplicon sequencing (48)
Primer design using Primer3Plus software
Size of amplicons (290 to 310 bp)
Removal of small DNA fragments using Agencourt AMPure XP
Amplicon quantification 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
Annotations
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