Massarray system

There were 19 common SNPs of HSPs and Rho GTPases selected in our study (Table 2). The candidate SNPs were located 5 kb upstream of the first exon and downstream of the last exon respectively. We used Haploview version 4.2 to choose the Haplotype tagging SNPs. They were chosen based on our previous research HSPs and Rho GTPases SNPs were related to lung cancer platinum-based chemotherapy toxicity (Zou et al., 2016 (link)), and they were associated with the outcome of cancers and involvement in multiple cancers (Li et al., 2016 (link); Hung et al., 2020 (link)). And the selected SNPs must meet the condition that the minor allele frequency (MAF)>0.05 in the HapMap CHB population. The DNA we used for genotyping was separated from a 5ml peripheral blood sample using FlexiGene DNA Kit (Qiagen, Hilden, Germany). And all the samples were stored at 4°C before using. Genotyping were conducted by Sequenom’s MassARRAY system (Sequenom, San Diego, California, USA).

To replicate the association results of the meta-analysis of the Asia array and the Fine Mapping array, we further analysed the 76 top variants in an additional 20,338 samples (6,369 cases and 13,969 controls, Supplementary Table 1) using the Sequenom MassARRAY system. All of these selected SNPs met the following quality criteria: (1) the MAF was higher than 0.5% in both the cases and controls; (2) HWE in the controls was P≥0.01 and the HWE in the cases was P>10⁻⁴; (3) SNPs with a meta-association of P<10⁻⁴ after adjustment for gender; (4) proximity to putative candidate genes (immune-related or involved in immune cell proliferation and differentiation) or known susceptibility loci for autoimmune diseases; and (5) in each locus, one or two of the most significant SNPs were selected for validation. For all of the 76 SNPs analysed in the validation study, the cluster patterns of the genotyping data from the Illumina and Sequenom analyses were checked to confirm their high quality. The genotype data are available in Table 2 and Supplementary Data 2.

Genomic DNA extracted from the samples was assayed for 30 single nucleotide polymorphisms (SNPs) associated with drug resistance using PCR-based single base extension on Sequenom MassARRAY system (Sequenom, San Diego, CA, USA) as previously described [20 ]. These includes the P. falciparum multidrug resistance gene (pfmdr1; 86, 184, 1034, 1042, 1246), the P. falciparum chloroquine resistance gene (pfcrt; 72, 76, 356, 371), the P. falciparum dihydrofolate reductase gene (pfdhfr; 16, 59, 108, 164), the P. falciparum dihydropteroate synthase gene (pfdhps; 463, 437, 581, 613), the P. falciparum cytochrome b gene (pfcytb; 268, 284, 356), and the P. falciparum multidrug resistance protein 1 gene (pfmrp1; 437, 876, 1390) The primers used in the analysis of the 30 SNPs are shown in S1 Table.

The EGFR T790M mutation was detected in the cells with matrix-assisted laser desorption/ionization time-of-flight MS using a standard protocol, on the MassARRAY System (Sequenom, San Diego, CA, USA). The mutant signal frequency was calculated as follows: mutant signal frequency (%) = (mutant peak height)/(mutant peak height + wild-type peak height) × 100.

There are multiple SNPs in MMP13 covering a ~13 kb region of chromosome 11q22.3. As a result, we utilized an htSNP approach to examine the MMP13 polymorphisms globally22 (link). Genotyped HapMap SNPs among Han Chinese population (HCB data, HapMap Rel 27, NCBI B36) with a minor allele frequency >5% were included in the selection. The htSNPs were chosen in a ~17 kb region (~13 kb MMP13 locus and 2 kb up-stream as well as 2 kb down-stream regions of MMP13). Four htSNPs were finally selected with Haploview 4.2 software on a block-by-block basis, using a method described previously with the sample size inflation factor, Rh², of ≥0.8 (Supplementary Table 2). MMP13 htSNPs (rs11225490, rs2252070, rs17099788 and rs3758854) were analyzed by the MassArray system (Sequenom Inc., San Diego, California, USA). A 5% blind, random sample of study subjects was genotyped in duplicates and the reproducibility was 99%.

Standard polymerase chain reaction (PCR) protocols were used to amplify the DNA specimens. Assay Design 3.1 software (Sequenom Inc., San Diego, CA, USA) was used to design the Primers for the PCR process. The PCR reaction was done in a total volume of 5 μL containing 1 μL DNA sample (10 ng/ μL), MgCl₂ 1.625 mM, 500 μM dNTP, PCR Buffer 1×, 1 unit HotStart Taq DNA polymerase, 0.1 μM PCR primers, and 1.8 μL ddH₂O. The PCR cycling started with an initial denaturation at 94 °C for 15 min, followed by 45 cycles of denaturation at 94 °C for 20 s, annealing at 56 °C for 30 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 3 min. SNP genotyping was determined by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) in the MassARRAY system (Sequenom, San, Diego, CA, USA). PCR reaction was performed on 384-well plates using a MassARRAY Nanodispenser (Sequenom). As quality control measures, the extracted DNA was checked for purity and concentration using ultraviolet spectrophotometer at ultraviolet (UV) readings of 260 nm and 280 nm (Beckman, USA). This was then followed by electrophoresis, PCR amplification, and cluster analysis with samples showing good scatter plots accepted for the final genotyping. Both blinded and unblinded samples were tested separately and replicated concordance rates of >99.99 %.