Typer 3

Procedures were preformed as previously described [Baldinger et al., 2014]. In short, 9 ml. EthyleneDiamineTetraacetic Acid (EDTA) blood samples were drawn from each subject and DNA was isolated from whole blood using the QiaAmp DNA blood maxi kit (Qiagen, Hilden, Germany). Genotyping was performed using the iPLEX assay on the MassARRAY MALDI‐TOF mass spectrometer as described [Oeth et al., 2009]. Allele specific extension products were identified and genotypes allocated by Typer 3.4 Software (Sequenom, San Diego, CA). All applied quality criteria were met [individual call rate >80%, SNP call rate >99%, identity of genotyped of CEU trios (Coriell Institute for Medical research, Camden, NJ) with HapMap database >99%].

Genotyping was conducted using the iPLEX Gold Platform (Sequenom, San Diego, CA) according to the manufacturer’s protocol. Briefly, iPLEX assays were scanned by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and individual SNP genotype calls were automatically generated with Sequenom TYPER 3.4 software. Genotyping was validated by the TaqMan allelic discrimination assay (Life Technologies, Carlsbad, CA). TaqMan genotyping was performed using a 7900HT Fast Real-Time PCR System (Life Technologies, Carlsbad, CA), with automated calls generated by SDS software based on discriminating plots (95% confidence). Genotyping was blind to case-control status and ethnic background of the samples.

Genotyping procedures were described in previous publications^{35 (link),46 (link)}. In summary, Ethylene-Diamine-Tetraacetic-Acid (EDTA) blood samples of 9 ml were extracted from each subject and whole blood was used for DNA isolation with QiaAmp DNA blood maxi kit (Qiagen, Hilden, Germany). The iPLEX assay and the MassARRAY MALDI‐TOF mass spectrometer were used for genotyping, for details please see^{47 (link)}. Identification of allele specific extension products and definition of genotypes was performed with Typer 3.4 Software (Sequenom, San Diego, CA). Quality requirements for genotyping were defined as an individual call rate above 80%, a SNP call rate over 99% and over 99% fit of genotyped CEU trios (Coriell Institute for Medical research, Camden, NJ) with the HapMap database.

Replication sample I was genotyped using the iPLEX assay on the MassARRAY MALDI‐TOF mass spectrometer as described (Oeth et al, 2009). Allele‐specific extension products were identified and genotypes allocated by Typer 3.4 Software (Sequenom, San Diego, CA, USA). All applied quality criteria were met (individual call rate > 80%, SNP call rate > 99%, identity of genotyped CEU Trios [Coriell Institute for Medical Research, Camden, NJ] with HapMap database > 99%).

All procedures were performed as previously described [13] . Briefly, DNA was isolated from peripheral blood mononuclear cells by the QIAamp DNA Mini-Kit (QIAGEN, Hilden, Germany). Genotyping of BDNF rs6265 single nucleotide polymorphism (SNP) was conducted with the MassARRAY platform (SEQUENOM, San Diego, CA) as described elsewhere [17] . PCR-primers were generated with the Assay Designer 4.0 software (SEQUENOM). Multiplex PCR reactions were performed with 12.5 ng of genomic DNA, 500 µM dNTPs (ABgene, Hamburg, Germany), 100 nM PCR primers, 1.625 mM MgCl2 and 0.5 U HotStar Taq polymerase (QIAGEN). Shrimp alkaline phosphatase (SAP) treatment, an iPLEX reaction cocktail with extension primers (7–14 µM), a iPLEX termination mix and an iPLEX enzyme (SEQUENOM) were added to the PCR-products. The resulting extension products were desalted using SpectroCLEAN resin (SEQUENOM), then spotted on SpectroCHIPs GenII (SEQUENOM) and analyzed with the MassARRAY MALDI-TOF mass spectrometer. Typer 3.4 Software was used to identify allele specific extension products and resulting genotypes (SEQUENOM). For genotyping quality assurance CEU HapMap Trios (Coriell Institute for Medical research, Camden, NJ) were included and compared with the HapMap-CEU population (www.hapmap.org). For all analyses val/val homozygotes ( = GG-carriers) were compared against met-carriers (AG- and AA-carriers).

Genotyping of TPH2 SNPs was performed using the iPLEX assay on the MassARRAY MALDI-TOF mass spectrometer as previously described by Oeth et al. [37 (link)]. Allele specific extension products were identified, and genotypes allocated by Typer 3.4 Software (Sequenom, San Diego, CA, USA). All applied quality criteria were met (individual call rate >80%, SNP call rate >99%, identity of genotyped of CEU Trios (Coriell Institute for Medical research, Camden, NJ) with 1000 Genomes database >99%). Here, rs1137070 and rs2064070 were in perfect LD, therefore statistical analyses were performed for rs1137070 only.

Blood samples were collected in tubes containing ethylene diaminetetraacetic acid (EDTA). After centrifugation at 1,500 rpm for 10 min, the samples were stored at −80°C. Genomic DNA from whole blood was extracted using the GoldMag DNA Purification Kit (GoldMag Co. Ltd, Xi'an City, China), and the purity and concentration was measured utilizing an ultraviolet spectrophotometer (Nanodrop, Thermo Scientific, Waltham, MA). We searched TLR1 gene SNPs in the NCBI dbSNP. Some of the SNPs were excluded due to the MAF were < 0.1 or the absence of genotype distribution data in Asian population. Eventually, combined with the current studies, two common polymorphisms (rs4833095 and rs5743557) in TLR1 gene were selected in the present study. We also calculated the statistical power of rs4833095, which was 0.86. This showed that our sample size was adequate and the results were acceptable. The Sequenom MassARRAY Assay Design 3.0 software was used to design Multiplexed SNP MassEXTEND assay. SNP genotyping was performed by using Sequenom MassARRAY RS1000 according to the standard protocol. The primers used for rs4833095 and rs5743557 are listed in Table 5. Sequenom Typer 3.0 software was used for data analysis.