Ion 520 chip kit

Targeted mutation analysis was performed by Next Generation Sequencing (NGS) (Ion GeneStudio S5 prime, Thermo Fisher Scientific, Waltham, MA, USA) using an AmpliSeq Custom Panel covering the most common recurrently mutated genes in papillary non-muscle invasive urothelial bladder cancer (complete coding sequence: ERCC2, FGFR3, PIK3CA, PTEN, STAG2 and TP53 (Supplementary Table S1). Amplicon library preparation and semiconductor sequencing was carried out according to the manufacturers’ manuals using the Ion AmpliSeq Library Kit v2.0, the Ion Library TaqMan Quantitation Kit, the Ion 510, Ion 520 and Ion 530 Kit—Chef and the Ion 520 Chip Kit (Thermo Fisher Scientific, Waltham, MA, USA). Five paraffin-embedded cases of papillary non-muscle invasive urothelial bladder served to validate the NGS panel. Variant calling of non-synonymous somatic variants compared to the human reference sequence was performed using Ion Reporter Software (Thermo Fisher Scientific, Waltham, MA, USA).

Targeted mutation analyses were performed by Next Generation Sequencing (NGS; Ion GeneStudio S5 prime, Thermo Fisher Scientific, Waltham, MA, USA) using an AmpliSeq custom panel designed for myeloid disorders comprising hotspot regions in 21 genes (JAK2, FLT3, STAT3, ASXL1, IDH1, IDH2, SRSF2, SF3B1, U2AF1, SETBP1, MPL, KIT, CBL, CSF3R, CALR, ETNK1, KRAS, NRAS, HRAS, BRAF, GNAS) and the 10 genes (CEBPA, RUNX1, IKZF1, DNMT3A, EZH2, ZRSR2, TP53, TET2, NPM1, STAG2). Amplicon library preparation and semiconductor sequencing was done according to the manufacturers’ manuals using the Ion AmpliSeq Library Kit v2.0, the Ion Library TaqMan Quantitation Kit, the Ion 510 & Ion 520 & Ion 530 Kit—Chef and the Ion 520 Chip Kit (Thermo Fisher Scientific, Fermont, CA, USA). Variant calling of non-synonymous somatic variants compared to the human reference sequence was performed using Ion Reporter Software (Thermo Fisher Scientific, Version 5.12.3.0). Variants were filtered with a threshold allele frequency of 5%.
Variants called by the Ion Reporter Software were visualized using the Integrative Genomics Viewer (IGV; Broad Institute, Cambridge, MA, USA; Version 2.5.2) to exclude panel-specific artefacts.

Genomic DNA was extracted from macro-dissected 5 µm paraffin sections using the Maxwell^® RSC DNA FFPE Kit and the Maxwell^® RSC Instrument (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
CTNNB1 exon 3 was investigated by next generation sequencing with a single amplicon using the Ion Amplicon Library Preparation Fusion Method (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol (gene specific primer sequences identical to Sanger sequencing: Fwd: 5’-TGGAACCAGACAGAAAAGCG-3’ and Rev: 5’-CAGGTACCGTGCGACATC-3’). Amplicons were purified and quantified applying Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA) and the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), respectively. Amplicons were diluted to 5 pM each and pooled. Clonal amplification and semiconductor sequencing was done according to the manufacturer’s manuals using the Ion 510™ and Ion 520™ and Ion 530™ Kit—Chef (ThermoFisher Scientific) on the Ion Chef™ Instrument (ThermoFisher Scientific) and the Ion 520™ Chip Kit on the Ion GeneStudio™ S5 Prime system (ThermoFisher Scientific). BAM Files were generated with Torrent Suite 5.10.2. Sequences were visualized and evaluated using the freely available software Integrative Genomics Viewer (IGV, Broad Institute).