Targeted amplicon sequencing

The sequencing libraries were prepared using a two-step PCR method targeting approximately 300 bp surrounding the somatic variants of interest. Primers targeting the genomic regions were designed using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) and forward adaptor sequence 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’ and reverse adaptor sequence 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’ were added to the primers necessary for the second round PCR amplification. The first round PCR utilized a gradient annealing temperature which decreased by 1 °C per cycle from 68 to 58 °C followed by 25 cycles with a 57 °C annealing temperature and 72 °C extension and 94 °C denature steps. Genomic (12.5 ng) DNA was used to amplify each of the target regions. After purification with AMPureXP beads (Beckman Coulter) and analysis on a Bioanalyzer DNA1000 chip (Agilent Technologies, Inc.) to ensure the desired target size, the amplicons from the same sample were normalized and pooled to a total of 15 ng using quantitation data from the Bioanalyzer. The amplicon pool from the first round PCR was amplified with eight cycles of PCR using a Nextera Index Kit (Illumina, Inc.), which uses primers that target the overhang adaptor sequence incorporated during the first round of PCR to add a unique indexed tag to each sample which allows pooling of libraries and multiplex sequencing. Indexed libraries were purified with AMPureXP beads, run on a Bioanalyzer DNA1000 chip to verify desired size distribution, quantitated using a KAPA qPCR library quantitation kit (Kapa Biosystems) and pooled in an equimolar fashion to a final concentration of 4 nM. 2 × 150 cycle sequencing was performed on a MiSeq (Illumina, Inc.), according to the manufacturer’s protocol.