Massarray typer analyzer software

Genotyping was performed using the iPLEX Gold Assay (Sequenom Inc.). Assays for all SNPs were designed using the eXTEND suite and MassARRAY Assay Design software version 3.1 (Sequenom Inc.). Amplification was performed in a total volume of 5 µL containing ∼10 ng genomic DNA, 100 nM of each PCR primer, 500 µM of each dNTP, 1.25× PCR buffer (Qiagen), 1.625 mM MgCl₂ and 1 U HotStar Taq (Qiagen). Reactions were heated to 94°C for 15 min followed by 45 cycles at 94°C for 20 s, 56°C for 30 s and 72°C for 1 min, then a final extension at 72°C for 3 min. Unincorporated dNTPs were SAP digested prior to iPLEX Gold allele specific extension with mass-modified ddNTPs using an iPLEX Gold reagent kit (Sequenom Inc.). SAP digestion and extension were performed according to the manufacturer's instructions with reaction extension primer concentrations adjusted to between 0.7–1.8 µM, dependent upon primer mass. Extension products were desalted and dispensed onto a SpectroCHIP using a MassARRAY Nanodispenser prior to MALDI-TOF analysis with a MassARRAY Analyzer Compact mass spectrometer. Genotypes were automatically assigned and manually confirmed using MassARRAY TyperAnalyzer software version 4.0 (Sequenom Inc.). The genotyped variants were then checked for concordance in allele frequencies with the exome sequencing data.

We searched for all SNPs in the ZPR1 region with minor allele frequencies (MAF) ≥0.01 using the dbSNP database (Build 143) of NCBI (Build 38). Using this strategy, 24 SNPs were identified (rs964184, rs11823543, rs17120029, rs11604424, rs1942478, rs139753514, rs12286037, rs4417316, rs12274192, rs6589566, rs7483863, rs2075290, rs618923, rs603446, rs76341142, rs11355367, rs74662600, rs10750096, rs3741298, rs140050044, rs12285095, rs2075294, rs33984246, and rs2266788). These 24 SNPs, which completely covered the region of ZPR1 (Fig. 1), were included in further analyses.
DNA was extracted from whole blood according to the standard protocol for the DNA Isolation Kit for Mammalian Blood (Tiangen Biotech Co., Ltd, Beijing, China). DNA was stored at −20 °C for SNP analyses. Genotyping was performed for all SNPs using the MassARRAY platform (Sequenom, San Diego, California, US). Briefly, the SNPs were genotyped using high-throughput, matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) mass spectrometry. Next, the resulting spectra were processed using the MassARRAY Typer Analyzer software (Sequenom, San Diego, California, US), and genotype data were generated from the samples. For quality control, both the case and control status were blind during all genotyping processes. Meanwhile, 5% of random samples were repeated, and the results were 100% concordant.

DNA was extracted from venous blood samples. We performed DNA genotyping using MassARRAY iPLEX platform (Sequenom Inc., San Diego, CA, USA), following the manufacturer’s protocol. We assessed SNP genotypes by MassARRAY Typer Analyzer software (v.4.0, Sequenom Inc., San Diego, CA, USA). The call rate for rs2925979 was above 95%. To verify reproducibility, blinded duplicates were selected randomly and run along with the samples. For Hardy–Weinberg equilibrium test, we randomly selected one sibling without T2DM from each family and found no violation for rs2925979 (p > 0.001).