Spectrochip array

In the current study, we focused on three genes (JAZF1, IGF1, and IGF2BP2) involved in lipid metabolism, which have been shown to affect insulin resistance 5 (link)-11 (link), 13 (link)-18 (link). The SNP rs864745 in JAZF1 is located in intron 1 28 (link), 29 (link) and rs35767 is located in the IGF1 promoter30 (link). The three SNPs (rs4376068, rs4402960, and rs6769511) of IGF2BP2 are all located in intron 2 6 (link), 31 (link).
The QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from peripheral lymphocytes. The aforementioned five SNPs in the three genes were genotyped using the MassArray Analyser 4.0 (Agena Bioscience, Inc., San Diego, CA, USA) as previously described 32 (link). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed using a device from Agena Bioscience. SNP genotyping was performed using a SpectroCHIP array, and the raw genotyping data were obtained using TYPER4.0 software (Agena Bioscience).

Mutational analysis was performed using the UltraSEEK™ Melanoma Panel v1.0 (Agena Bioscience, Hamburg, Germany), interrogating 61 clinically relevant variants across 13 genes, including BRAF, NRAS, KIT and MAP2K1, detected at as low as 0.1% minor allele frequency. Reactions were performed as described before [46 (link)]. In brief, PCR (45 cycles) was followed by shrimp alkaline phosphatase treatment and single base primer extension, using biotinylated ddNTPs specific for the mutant alleles. After capture of the extended primers using streptavidin-coated magnetic beads, a cation-exchange resin was added for cleaning, and 10–15 nL of the reaction was transferred to a SpectroCHIP^® Array (a silicon chip with pre-spotted matrix crystals) using an RS1000 Nanodispenser (Agena Bioscience). Data were acquired via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, using a MassARRAY Analyzer 4 (Agena Bioscience, Hamburg, Germany). After data processing, a spectrum was produced with relative intensity on the y-axis and mass/charge on the x-axis. Typer Analyzer software was used for data analysis and automated report generation. Sanger sequencing was performed to verify mutations detected by the UltraSEEK™ Melanoma Panel, and only mutations which were detected in both assays (98%) were used for further analysis.

UltraSEEK assays were designed using the AgenaCx online assay design software which automatically selects the PCR and extension primers (Supplementary Table 4), and adds to each reaction control assays for PCR and capturing. All oligonucleotides were obtained from Integrated DNA Technologies and control oligos from Agena Bioscience GmbH. Reactions were performed as described before^{36 (link)}, using reagents obtained from Agena Bioscience. Briefly, PCR (45 cycles) was followed by shrimp alkaline phosphatase treatment and single base primer extension using biotinylated ddNTPs specific for the mutant alleles. After capture of the extended primers using streptavidin-coated magnetic beads, a cation-exchange resin was added for cleaning and 10-15 nl of the reaction was transferred to a SpectroCHIP® Array (a silicon chip with pre-spotted matrix crystals) using an RS1000 Nanodispenser (Agena Bioscience). Data were acquired via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a MassARRAY Analyzer 4 (Agena Bioscience). After data processing, a spectrum was produced with relative intensity on the y-axis and mass/charge on the x-axis. Typer Analyzer software was used for data analysis and report generation.