We attempted validation of all de novo coding variants predicted in TD subjects. We PCR-amplified whole-blood derived DNA and then Sanger sequenced the amplicon. We assessed all family members to ensure that (1) the variant was present in the child, and (2) absent in both parents. We predicted 301 de novo coding variants in the 311 TIC Genetics trios; however, we were unable to attempt confirmations on 51 of these variants due to difficulties with primer design, PCR amplification, and/or Sanger sequence quality. In the 250 variants with confirmation data, 243 confirmed as true de novos (97.2%). 240/245 of the de novo SNVs confirmed (97.8% confirmation rate), and 3/5 of the de novo indels confirmed (60%).
