Iplex gold reagent kit

All putative de novo mutations were tested by both Sanger sequencing and the MassARRAY system (Agena Bioscience, San Diego, CA). For assays with the MassARRAY system, PCR amplification and single base extension primers for each mutation were designed using Assay Design Suite software (Agena Bioscience), and PCR amplification and single base extension reactions were performed using reagents supplied in the iPLEX Gold reagent kit (Agena Bioscience). To evaluate background noise, PCR reactions containing no template DNA were also performed. MassARRAY data were analyzed using a Typer Analyzer (Agena Bioscience), which facilitates visualization of the raw spectra and determines the degree of confidence for wild-type and mutant peak signals. A true mutant was defined as having a peak probability of at least 0.95. For assays with Sanger sequencing, the same PCR amplification primers for MassARRAY assays were used. The Sanger sequencing chromatograms were visualized using Chromas V2.6.6 (https://technelysium.com.au/wp/). The presence or absence of a mutation was determined manually by whether there is a mutant peak at the target site.