Ion Torrent Next Generation Sequencing Analysis

Primers for genes involved in NFPanNETs (MEN1, ATRX, DAXX, VHL) were designed for a custom Ampliseq Panel using Life Technologies software, Ion Ampliseq Designer. Ampliseq libraries were prepared from DNA samples extracted from NFPanNET samples using the RNA/DNA/Protein Purification Plus Kit (47700, Norgen Biotek Corp., Thorold, ON, Canada). DNA sample concentration were determined using Nanodrop (Thermo Fisher’s ND8000, Waltham, MA). Final sample qualification and quantitation was established with Picogreen analysis (standard curve assay) on a Molecular Devices Gemini XPS Plate Reader and Softmax Pro Software (Sunnyvale, CA). All samples and controls were run in triplicate.

Ampliseq libraries were prepared for each DNA sample. To create libraries, each DNA sample was first amplified according to the manufacturer’s protocol. Each library was then assigned a unique barcode-adapter sequence and ligated onto the libraries. The final libraries were qualified and quantitated using Life Technologies’ Ion Library Quantitation Kit (Item #4468802). Libraries were then cycled and analyzed on an ABI PRISM^® 7900HT Sequence Detection System. Using the quantitation values from the real time PCR analysis, all libraries were then normalized with 1X Low TE Buffer to either 50pMol or 100pMol to prepare for emulsion PCR. Libraries were then combined into pools of 9 for a total of 4 pools. Combined with an Ion 318 chip, roughly 300X coverage per amplicon was achieved. The clonal emulsion amplification of Ampliseq libraries was sequenced on the Ion Torrent PGM platform, using Life Technologies OT2 system (Waltham, MA). The OT2 system is automated, and when used in conjunction with the Enrichment Station (ES), generates template positive Ion PGM Template One Touch 2 200 Ion Sphere Particles for semiconductor sequencing. All analysis and data QC was completed with Life Technologies Ion Torrent Suite v5.0.4.