title: Sequening using Thermo Fisher Cancer Hotspot v2 probes of bulk brain: hippocampus tissue from subject FC8401 for the temporal lobe epilepsy (TLE) project.
design: Hippocampus derived DNA was sequenced with a custom gene-panel (Starting from recurrent cancer hotspot mutations in a commercially-available panel (Cancer Hotspot v2, Thermo Fisher) and cancer-linked mutations in MTOR-pathway genes (COSMIC, accessed Nov. 2018), we selected gain-of-function SNPs, prioritizing variants occurring multiple times in COSMIC datasets and submitting top variants for Ampliseq pool design with manufacturer-designed software (Ampliseq, Thermo Fisher)). To generate Ampliseq libraries, 10 ng of genomic DNA was added to 1X final Phusion U Hot Start Master Mix (Thermo Fisher), 0.9X Ampliseq primer pool, and 80 ng ET SSB (NEB), undergoing 17 cycles of PCR before standard Illumina library preparation. The panel was sequenced on a Nextseq 500 high output flow cell.