Targeted Loci Amplification and Sequencing

PCR reactions to amplify targeted loci were performed using the primers shown in Supplementary Table 5. For most loci, we were able to use standard PCR conditions with Phusion Hot Start II high-fidelity DNA polymerase (Thermo-Fisher) performed according to manufacturer’s instructions for 35 cycles (98°C, 10 s denaturation; 68°C, 15 s annealing; 72°C, 30 s extension). For loci that did not amplify under standard conditions we used one of the following modifications: 1) the addition of betaine to a final concentration of 1.8M, 2) touchdown PCR ([98°C, 10 s; 72–62°C, −1°C/cycle, 15s; 72°C, 30s]_{10 cycles}, [98°C, 10 s; 62°C, −1°C/cycle, 15s; 72°C, 30s]_{25 cycles}) with 1.8M betaine, and 3) the addition of 3% or 5% DMSO and an annealing temperature of 65°C. PCR products were analyzed for correct size on a QIAxcel capillary electrophoresis system. Correctly sized products were treated with ExoSap-IT (Affymetrix) to remove unincorporated nucleotides or primers and sent for DNA sequencing to confirm the endogenous gene sequence.