Massarray snp genotyping system

The newly identified 12 mutations in our recent studies, including the c.1470G>T in BMPR1B [7 (link)]; the g.46547859C>T, c.1040T>C, and g.46544883A>G in GDF9 [5 (link)]; the g.46547645T>G in GDF9 [6 ]; the g.509807863G>A in BMP15 [8 (link)]; the c.240C>T and c.279C>T in LEPR [9 (link)]; and the g.25929637G>A, g.25929679T>C, g.25929819A>G, and g.25929965A>T in B4GALNT2 [10 ], as well as seven known mutations including the c.746A>G (FecB) in BMPR1B [11 (link),12 (link),13 (link)]; the c.260G>A (FecG¹) [14 (link)] and c.994A>G (FecG^A) in GDF9 [14 (link)]; the c.31_33CTTinsdel (B1) and c.755T>C (Lue252Pro) in BMP15 [14 (link),15 (link)]; the c.185G>A (FecD) in LEPR [16 (link)]; and the c.440C>T and c.823C>T in B4GALNT2 [17 (link)], were genotyped with the MassARRAY^® SNP genotyping system (Agena Bioscience, San Diego, CA, USA) in the 325 UM, 30 DPU, 66 SFKU, 184 SN, 30 Tan, 30 Hu, and 30 STH sheep populations. Assay Design Suite (http://agenabio.com/assay-design-suite-20-software, 24 June 2022) was used to design PCR and extension primers from the sequences, including each target mutation and around 100 upstream and downstream bases. With the help of the Sequenom MassARRAY iPLEX technology, the genotype of each SNP was examined [18 (link)]. The software MassARRAY Typer 4.0 Analyzer (Agena Bioscience) was used to examine the obtained data.

The FecX^R, FecX^Gr, FecX^O, and FecX^Bar in BMP15, and the FecG^T, FecG^V, FecG^A, and FecG¹ in GDF9 genes, were genotyped with the MassARRAY^® SNP genotyping system (Agena Bioscience, San Diego, CA, USA) in the 260 MG population. PCR and extension primers were designed from sequences containing each target mutation and ~100 upstream and downstream bases with Assay Design Suite (http://agenabio.com/assay-design-suite-20-software) using the default settings. The genotype of each allele was analyzed using the Sequenom MassARRAY iPLEX platform [37 (link)]. The resulting data were analyzed using the MassARRAY Typer 4.0 Analyzer software (Agena Bioscience, San Diego, CA, USA).

SNP genotyping of the case group was performed using the MassARRAY SNP genotyping system (Agena Bioscience San Diego, EUA) according to manufacturer’s instructions at the National Genotyping Center (CEGEN), on a panel of 45 SNP assays. The primers for amplification and extension were designed using the Extend Primer Assay Design software v4. Sequenom iPLEX GOLD chemistry was used for locus-specific amplification, followed by a single-base primer extension reaction, which generated products of different masses that were quantitatively analyzed using MALDI-TOF mass spectrometry. The resulting data were analyzed using TyperAnalyzer software v 4, followed by manual inspection of the spectra by trained personnel [23 ]. All assays were performed in 384-well plates, including negative controls and a trio of Coriell samples (Na10830, Na10831, and Na12147) for quality control.