Fig. 3

Schematic overview of the smMIP assay. (a) First, the single molecule molecular inversion probe (smMIP) capture procedure is performed. smMIPs are long oligonucleotides consisting of two targeting arms (extension probe and ligation probe), joined by a backbone. The probe sequences are complementary to genomic DNA sequences surrounding the target region that covers a hotspot location (indicated by the yellow asterisk). During the capture reaction, smMIPs are hybridized to genomic DNA (gDNA), followed by an extension and ligation reaction, which results in circular smMIPs. Subsequent exonuclease treatment will remove all linear gDNA and unused smMIPs. Between the backbone and probe sequences are primer sequences (green bars) that are used to amplify the target region, followed by library preparation and next-generation sequencing (NGS). (b) By including a single-molecule tag of 8 random nucleotides (N₈) at the end of the ligation probe, duplicate reads can be identified and merged into a consensus thereby removing PCR and sequencing artifacts. Genuine C > T and G > A mutations can be distinguished from deamination artifacts by strand specific amplification of the smMIPs. In our smMIP design, exon 8 and exon 9 of the GNAS gene are each covered by two independent smMIPs targeting both strands (smMIP1–2 and smMIP3–4, respectively)