KIT mutations in primary mediastinal B-cell lymphoma

Supplemental Methods. Library preparation and semiconductor sequencing. For screening of the three PMBL cell lines we employed a multiplex PCR based Ion Torrent AmpliSeqTM cancer panel (Life Technologies) covering approx. 2,800 COSMIC annotated mutations in 50 key cancer genes (Supplemental Table 1). Amplicon library preparation was performed using ~10ng of DNA as advised by the manufacturer. Briefly, the DNA was mixed with the primer pool, containing all primers for generating the 207 amplicons, and the AmpliSeq HiFi Master Mix and transferred to a PCR cycler (BioRad, Munich, Germany). PCR cycling conditions were as follows: Initial denaturation: 99°C for 2 min, cycling: 21 cycles of 99°C, 15 sec and 60°C, 4 min. After the end of the PCR reaction, primer end sequences were partially digested using FuPa reagent as instructed, followed by the ligation of barcoded sequencing adapters (Ion Xpress Barcode Adapters, Life Technologies). The final library was purified using AMPure XP magnetic beads (Beckman Coulter, Krefeld, Germany) and quantified using qPCR (Ion Library Quantitation Kit, Life Technologies) on a StepOneqPCR machine (Life Technologies). The individual libraries were diluted to a final concentration of 20pM and eight libraries were pooled and processed to library amplification on Ion Spheres using Ion OneTouch 200bp chemistry. Unenriched libraries were quality-controlled using Ion Sphere quality control measurement on a QuBit instrument. Best results were obtained by lowering the recommended library input to 4µl (20pM), as this reduced the rate of polyclonal beads. After library enrichment (Ion OneTouch ES), the library was processed for sequencing using the Ion Torrent 200bp sequencing chemistry and the barcoded eight libraries were loaded onto a single 318 chip. Data analysis. Raw data analysis was performed using Ion Torrent Software Suite (Version 2.2 and 3.0). The reads were aligned to the human reference sequence build 38 (hg19) using the TMAP aligner implemented in the Torrent Suite software or alternatively using a commercial third party software (CLC genomics Suite 5.5, CLCbio, Aarhus, Denmark). Detection of single base pair variants and insertion-deletion polymorphisms (InDels) compared to the human reference sequence was performed using either Ion Torrent Variant Caller (2.2 and 3.0) or the two build-in variant detection algorithms of the CLC Genomics suite 5.5 (probabilistic or quality-based variant caller). Detection thresholds for SNPs and InDels were set at an allele frequency of 5%. Analysis of KIT-Mutation Status in Primary Patients Samples. Tumor areas were micro dissected and subsequently lysated with proteinase K according …