Massarray platform

Genotypes were determined on whole-genome amplified DNA (primer extension pre-amplification) by the Agena Bioscience MassArray platform (formerly SEQUENOM) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described
^{25 (link)}. Details of the primer sequences and assays are given in
Extended datafiles 1 and
2^{29 (link)}. The most informative haplotype-tagging SNPs (htSNPs) to type in Gambian subjects were identified by analysing the pattern of linkage disequilibrium (LD) in the
IFNG gene loci using previously available data from 32 Gambian family trios
^{25 (link),
30 (link)}. The PHASE program (
http://stephenslab.uchicago.edu/software.html) version 2.1 was used to infer haplotypes from the genotypes of the study population and estimate the frequency of each inferred haplotype
^{31 (link)}. The entropy maximization method was used to identify htSNPs that described >90% of the observed haplotypic diversity in this gene region
^{30 (link)}. The HaploXT program (
http://www.sph.umich.edu/csg/abecasis/GOLD/docs/haploxt.html) was used to estimate pairwise LD statistics. Sickle cell (HbS, rs334) and glucose-6-phosphate deficiency (G6PD) deficiency (rs1050828 and rs1050829) were also genotyped using the Agena Bioscience MassArray platform.

Based on previous studies [20 (link), 21 (link)], 1000 Genomes Chinese Han Beijing population and dbSNP database (https://www.ncbi.nlm.nih.gov/snp/) with a minor allele frequency (MAF) > 0.01, and Hardy–Weinberg equilibrium (HWE) > 0.05, we selected rs2069837 and rs13306435 in IL-6 gene for genotyping. Genomic DNA was extracted from peripheral blood samples using the GoldMag DNA Extraction Kit (GoldMag Co. Ltd, Xi’an, China). The concentration and purity of DNA were assessed using the NanDrop 2000 (Thermo Scientific, USA).
The primer sequence of rs2069837 and rs13306435 was presented in Additional file 1: Table S1. PCR reactions were performed in a buffer containing 1 μl DNA, 0.5 μl PCR Buffer, 0.4 μl MgCl₂, 0.1 μl dNTP Mix, 1.0 μl primer mix, and 0.2 μl Taq ligase in a final reaction volume of 5 μl. The reaction mixture was heated to 94 °C for 15 min for denaturation. Then, the sample was subjected to 45 cycles of 94 °C 20 s, annealing at 56 °C 30 s and extension at 72 °C 60 s, followed by a final extension step at 72 °C for 3 min. The PCR product was used to genotype using the Agena MassArray platform (Agena Bioscience, San Diego, CA, USA) [21 (link), 22 (link)]. Then, the raw data was analyzed and managed using the Agena Typer 4.0 software.

Genomic DNA extraction was performed on the basis of the manufacturer’s procedures of GoldMag Beads DNA Extraction Kit (GoldMag, Xi’an, Shaanxi, China). DNA concentration was determined by Spectrometry (Beckman Instruments, Fullerton, CA, USA). Agena MassARRAY platform (Agena Bioscience, San Diego, CA, USA) was utilized for SNP genotyping. The design of extended primers was conducted by the Agena online design software (https://agenacx.com/online-tools/), and the sequences were listed in Supplementary Table 1. The process of genotyping was double-blinded by two laboratory personnel. For quality control, 10% of the samples were randomly chosen for repeated genotyping, and the reproducibility was 100%.
All seven candidate SNPs in CYP19A1 (rs4646, rs6493487, rs1062033, rs17601876 and rs3751599) and CYP1A2 (rs762551 and rs2470890) were screened using the database of dbSNP in NCBI and the 1000 Genomes Project data, and the selection criteria were as follows: i) the minor allele frequency (MAF) of all SNPs was greater than 5%; ii) call rate was over 95% during genotyping; iii) r², a pairwise linkage disequilibrium (LD), was over 0.8. Besides, we applied RegulomeDB annotations to predict the effect of these SNPs according to the rank score evaluated by a model integrating functional genomic features.