Ampliseq exome kit

We performed WES using an Ion Proton System equipped with a PI chip V2 together with an AmpliSeq Exome kit (Thermo Fisher Scientific) [26 (link)]. Briefly, 100 ng each of tumor and matched blood cell DNA was used for target amplification with the following protocol: 99 °C for 2 min, followed by 10 cycles at 95 °C for 15 s and 60 °C for 16 min, and a final hold at 10 °C. The incorporated primer sequences were partially digested using FuPa Reagent (Thermo Fisher Scientific). Ion Torrent Proton adapters were ligated to the amplicons at 22 °C for 30 min and then at 72 °C for 10 min. The amplicon library was purified using Agencourt AMPure XP Beads (Thermo Fisher Scientific). The library DNA was quantified by qRT-PCR, and 7 pM library DNA was used for sequencing. The sequencing data were aligned to the human reference genome (assembly GRCh37/hg19) and were quality trimmed using Ion Torrent Suite version 4.2 (Thermo Fisher Scientific). The mutations were visualized using the Integrative Genomics Viewer [27 (link)] and were validated using Sanger sequencing or pyrosequencing.

At the first stage, exome sequencing was performed on 40 randomly selected probands from the MODY group. The exome sequencing was carried out on an Illumina HiSeq 1500 instrument (Illumina, San Diego, CA, USA). The enrichment and library preparation were performed using the SureSelectXT Human All Exon v.5 + UTRs Kit. Whole-exome libraries were prepared with the AmpliSeq Exome Kit (Thermo Fisher Scientific, Walthamm, MA, USA). On the DNA samples from the other patients (138 unrelated probands), we performed targeted sequencing. In the target panel, we included coding parts and adjacent splicing sites of the following MODY-associated genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, KCNJ11, ABCC8, and APPL1. Oligodeoxynucleotide probes and the KAPA HyperPlus Kit (Roche, Basel, Switzerland) were used to prepare libraries at the Genomics Multi-Access Center (Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia). The quality of the analyzed DNA and of the prepared libraries was evaluated by means of a capillary electrophoresis system, the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA). Analysis of each fully prepared library was conducted on the Illumina MiSeq platform (Illumina, San Diego, CA, USA).