Targeted NGS Analysis of Cancer Genes

For targeted NGS analysis, Ion AmpliSeq designer software (Life Technologies, Tokyo, Japan) was used to design primers, which consisted of 610 primer pairs in two pools covering the exons and exon–intron boundaries of 25 cancer-predisposing genes (APC, ATM, BARD1, BMPR1A, BRIP1, CDH1, CDK4, CDKN2A, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, SMAD4, STK11, and TP53) (Walsh et al. 2011 (link); Couch et al. 2014b (link); Economopoulou et al. 2015 (link); Tung et al. 2015 (link)). Multiplex polymerase chain reaction (PCR) was performed using 50–100 ng genomic DNA with 17 cycles with a premixed primer pool using Ion AmpliSeq Library Kit 2.0, as previously described (Hirotsu et al. 2014 (link)). The PCR amplicons were treated with 2 μL FuPa reagent to partially digest primer sequences and phosphorylate the amplicons. The amplicons were ligated to adapters with the diluted barcodes of the Ion Xpress Barcode Adapters kit (Life Technologies). Adaptor-ligated amplicon libraries were purified using Agencourt AMPure XP reagents (Beckman Coulter, Tokyo, Japan). The library concentration was determined using an Ion Library Quantitation Kit (Life Technologies), then each library was diluted to 8 pM and the same amount of libraries was pooled for one sequence reaction. Next, emulsion PCR was carried out using the Ion OneTouch System and Ion PI Template OT2 200 Kit v2 (Life Technologies) according to the manufacturer’s instructions. Template-positive Ion Sphere Particles were then enriched with Dynabeads MyOne Streptavidin C1 Beads (Life Technologies) using an Ion OneTouch ES system (Life Technologies). Purified Ion Sphere particles were loaded on an Ion PI Chip v2. Massively parallel sequencing was carried out on an Ion Proton System (Life Technologies) using the Ion PI Sequencing 200 Kit v2. Sequencing was performed using 500 flow runs that generated ∼200 bp reads.