Massarray assay design 3

Target gene sequences of the pathogens were downloaded from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/), and at least 300 sequences were obtained for each pathogen to ensure that they were representative. MAFFT was used to align multiple sequences and select highly homologous sequences as candidates for primer design (https://www.ebi.ac.uk/Tools/msa/mafft/). Primers and single-base extension primers for multiplexed assays were designed using the MassARRAY Assay Design 3.1 software (Agena Bioscience, Inc., San Diego, CA, USA) (Table 3). GAPDH was used as an internal control, and the target specificity of the primers and probes was checked using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All primers were synthesized by BGI Tech Solutions (Beijing Liuhe Greatness Technology Co., Ltd., Beijing, China).

Due to the high similarity of SMN1 and SMN2 genes, two well-known positions (c.840 and c.1155) were used as targets for designing PCR and single-base extension (SBE) primers (Figure 1). The iPLEX assay consists of a target-specific PCR reaction, followed by SBE using molecular weight-modified dideoxynucleotide terminators of an extension primer which anneals immediately upstream of the polymorphic site of interest. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the distinct mass of the extended primer identifies the SNP allele. The nucleotide of c.840 position is C and T in SMN1 and SMN2 genes, respectively. The nucleotide of c.1155 position is G and A in SMN1 and SMN2 genes, respectively. PCR and SBE primers (patent applications in progress) were designed for these two variants of SMN1 and SMN2 genes by using MassARRAY Assay Design 3.1 software (Agena, San Diego, CA) with 80 ≤ amplicon length (bp) ≤120 and 4,300 ≤ Mass Range (Da) ≤ 9,400. One PCR primer pair was designed for amplifying exon 7 of these two genes, and another was for exon 8. Beside these two variants of SMN1 and SMN2 genes, we also designed other polymorphic markers for sample identification (data not shown).

The 56 SNPs were genotyped in 191 LN patients and 171 SLE without LN patients by using the Agena MassARRAY system. Agena MassARRAY Assay Design 3.1 software was used to design a multiplexed SNP MassEXTEND assay, and SNP genotyping was performed using Agena MassARRAY RS1000 with the manufacturer’s protocols. Agena Typer 4.0 software was employed to perform data management and analyses. Three cases with call rates <90% were excluded. Two SNPs were excluded because of a call rate <90% or a P value of Hardy-Weinberg equilibrium <0.001. The remaining SNP cluster patterns from the genotyping data were checked to confirm their good quality. After quality control, 54 SNPs in 188 LN patients and 171 SLE without LN patients were remained in the replication stage analysis.

PCR and iPLEX extension primers were designed for 17 SLC22A5 mutations using MassArray Assay Design 3.1 software (Agena, San Diego, CA),with amplicon lengths of 80–120 bp and a mass range of 4,500–9,000 Da. The designed primers were run through BLAT and modified when necessary to avoid pseudogene amplification. The designed primers cover nearly all the most common SLC22A5 gene mutation sites, based on the mutation frequencies reported in previous studies [5 (link), 9 , 24 (link), 26 (link), 28 , 29 (link)] and a local mutation database (Additional file 1: Table S1). Genotyping was performed using the MassARRAY platform according to the manufacturer’s instructions.

Six SNPs (rs2243250, rs2227284, rs2243267, rs2243270, rs2243283, rs2243289) with a minor allele frequency (MAF) > 0.05 in IL‐4 gene (its GenBank reference is NC_000005.10) were selected from theHapMap database. Genomic DNA was extracted from blood sample by GoldMag‐Mini Whole Blood Genomic DNA Purification Kit (GoldMag Ltd. Xi'an. China) according to manufacturer's protocol, and the concentration of DNA was measured by Nanodrop 2000. MassARRAY Assay Design 3.0 Software (Agena, San Diego, CA, USA) was utilized to design Multiplex SNP MassEXTEND assays (Yang et al., 2005). SNPs genotyping were performed based on Agena MassARRAY RS1000 (Agena, Inc.) according to the manufacturer's protocol, and the Agena Typer Software, version 4.0 (Agena, Inc.) was used to manage and analyze the data as earlier described (Gabriel, Ziaugra, & Tabbaa, 2009; Thomas et al., 2007).