Sanger Sequencing of Candidate Variants

PCR amplification of selected candidate variants from exome sequence analysis were amplified using standard PCR primers. Amplicons were assessed via agarose gel electrophoresis, then purified by treating 5 μl of PCR product with 2μl of Exonuclease and Shrimp Alkaline Phosphatase (Exo-SAP-IT; Affymetrix) and submitted to the Molecular Genetics Core Facility at Boston Children’s Hospital or the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for sequencing using the ABI Prism BigDye Terminator cycle sequencing protocols (Applied Biosystems, Perkin-Elmer Corp., Foster City, CA). Sequence data were generated in an ABI Prism® 3130 or 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA), formatted by ABI Sequencing Analysis software v.5.2 and KB Basecaller, and analyzed using Sequencher v.5.2.3 or earlier versions (GeneCodes Corporation, Ann Arbor, MI). Sanger sequencing was performed in affected family members and other informative family members to confirm pathogenic mutations and track co-segregation patterns. The only widespread screening performed via Sanger sequencing was for FKRP in 18 families who had exome sequencing on an older platform that did not have good coverage of that gene^{7 (link)}.