Massarray typer software 4

The tagSNPs covering the HOTAIR region were selected from 1000 genome CHB data (phase 3, minor allele frequency ≥ 5%, pairwise r2 ≥ 0.8) using the Haploview 4.2 software. We also considered the potential functional variants that were located in the binding site of miRNAs. Finally, six SNPs, including rs4759314, rs12427129, as well as four SNPs in 3′UTR of HOTAIR gene (rs200349340, rs12312094, rs1838169, and rs7958904), were finally selected as the candidates (Fig. 1). Genotyping was conducted using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (the mass spectrum plots for three genotypes of rs200349340 were presented in Additional file 1: Figures S1–S3). Genotype calling was conducted using MassARRAY RT software version 3.0. Genotyping data was then analyzed by MassARRAY Typer software 4.0.3 (Sequenom, San Diego, CA, USA) in conjunction with Sequenom’s iPLEX™ Gold assay. Quality control was detected using Sanger sequencing of ~ 10% randomly selected samples, yielding a 100% concordance.Fig. 1

Relative location about the 6 candidate SNPs in HOTAIR gene

We selected 4 SNPs by analyzing GFAP-related Han Chinese data from 1000 Genome Project resources (http://www.1000genomes.org). The candidate SNPs should meet these criteria: (1) the minor allele frequency (MAF) > 0.05; (2) r² < 0.80; (3) at the 3'UTR of GFAP gene. The significant SNP rs11558961 is located at a miR-139 binding site, with predicted proximal transcriptional regulatory potential (http://rsnp.psych.ac.cn/) (http://snpinfo.niehs.nih.gov/cgi-bin/snpinfo/snpfunc.cgi). rs1042329, rs8067254, and rs17027 were also predicted as functional SNPs, which were seated nearby other miRNA binding sites.
All SNPs of GFAP gene were genotyped using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Amplification and single-base extension primers applied in multiple PCR were synthesized by Benegene (Benegene Biotechnology, Shanghai, China) (Table 3). The product of each sample was dispensed onto a 384-format SpectroCHIP with the MassARRAY Nanodispenser RS 1000. Then the MALDI-TOF MS assay was performed on a MassARRAY Compact Analyzer. Genotype calling was conducted using MassARRAY RT software version 3.0. Data was analyzed by MassARRAY Typer software 4.0.3 (Sequenom, San Diego, CA, USA). Genotyping quality was detected using Sanger sequencing of ~10% randomly selected samples, yielding a 100% concordance.