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27 protocols using MassARRAY iPLEX platform

To genotype the EBV variants in the 990 cases and 1105 controls from
Zhaoqing, the customized primers and the protocol recommended by the Agena
Bioscience MassArray iPLEX platform were used. A fixed position in the human
albumin gene was used as a positive control. Because the genotyping success rate
strongly correlates with the EBV DNA abundance (Supplementary Fig. 15), about half
of the validation samples (483 of the cases and 605 of the controls) could be
successfully genotyped for all the three GWAS candidate markers (i.e., SNPs
162215C>A, 162476T>C and 163364C>T). The slightly lower
success rate in the cases is consistent with the fact that the EBV DNA abundance
was lower in the saliva from patients than from controls. For detailed
information, see Supplementary
Note
.
Seven previously reported human SNPs in HLA (rs2860580,
rs2894207 and rs28421666), CDKN2A/2B (rs1412829),
TNFRSF19 (rs9510787), TERT (rs31489) and
MECOM (rs6774494) were genotyped using customized primers
and following the protocol recommended by the Agena Bioscience MassArray iPLEX
platform in the 990 cases and 1105 controls from Zhaoqing. A fixed position in
the human albumin gene was used as a positive control. The genotyping completion
rate for all seven human SNPs was > 95%. Associations with NPC were
assessed with logistic regression under an additive model adjusted for sex and
age.
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To genotype the EBV variants in the 990 cases and 1105 controls from
Zhaoqing, the customized primers and the protocol recommended by the Agena
Bioscience MassArray iPLEX platform were used. A fixed position in the human
albumin gene was used as a positive control. Because the genotyping success rate
strongly correlates with the EBV DNA abundance (Supplementary Fig. 15), about half
of the validation samples (483 of the cases and 605 of the controls) could be
successfully genotyped for all the three GWAS candidate markers (i.e., SNPs
162215C>A, 162476T>C and 163364C>T). The slightly lower
success rate in the cases is consistent with the fact that the EBV DNA abundance
was lower in the saliva from patients than from controls. For detailed
information, see Supplementary
Note
.
Seven previously reported human SNPs in HLA (rs2860580,
rs2894207 and rs28421666), CDKN2A/2B (rs1412829),
TNFRSF19 (rs9510787), TERT (rs31489) and
MECOM (rs6774494) were genotyped using customized primers
and following the protocol recommended by the Agena Bioscience MassArray iPLEX
platform in the 990 cases and 1105 controls from Zhaoqing. A fixed position in
the human albumin gene was used as a positive control. The genotyping completion
rate for all seven human SNPs was > 95%. Associations with NPC were
assessed with logistic regression under an additive model adjusted for sex and
age.
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In this study, six SNPs (rs10998449, rs10762244, rs10998461, rs10998468, rs7903209, and rs4472827) in the STOX1 were selected from the DbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) and 1,000 genome (http://www.internationalgenome.org/). All the SNPs were selected at a minor allele frequency >5% in Han Chinese from the 1,000 Genome Projects.
According to the manufacturer's protocol, GoldMag‐Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd.) was used to isolate the total genomic DNA from peripheral blood. The Agena Bioscience Assay Design Suite V2.0 software (http://agenacx.com/online-tools) was used to design the extended primer. The MassARRAY Nanodispenser (Agena Bioscience) and MassARRAY iPLEX platform (Agena Bioscience) were used to genotype, and the Agena Bioscience TYPER software (version 4.0) was used to analyze the data. We randomly selected about 10% of the sample to repeat genotyping, and the reproducibility was 100% indicating that our result is reliable.
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We selected 47 SNPs from 21 loci based on evidence they were low-penetrant risk variants for melanoma in other studies (Amos et al., 2011 (link), Barrett et al., 2011 (link), Bishop et al., 2009 (link), Fernandez et al., 2008 (link), Gudbjartsson et al., 2008 (link), Han et al., 2008 (link), Jannot et al., 2005 (link), Law et al., 2012 (link), Macgregor et al., 2011 (link), Nan et al., 2011 (link), Zhang et al., 2012 (link)). DNA was extracted from buccal swab kits (Begg et al., 2005 (link)). SNPs were genotyped using the MassArray iPLEX platform (Agena Bioscience) with quality control measures described previously (Orlow et al., 2012 (link)). CASP8 rs10931936 and ATM rs1801516 (Barrett et al., 2011 (link)) were not compatible with the platform design, and proxy SNPs rs6735656 and rs12278954, respectively, (r2 > 0.95) were chosen (1000 Genomes, CEU population; Proxy SNP; Broad Institute).
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Genotype data for the TW1 cases cohorts, the NZ, and US cases cohorts and NZ controls cohort were accessed from previously published studies29 (link),32 (link),36 (link),38 (link). SNP genotyping of TW1, NZ and US cases and controls cohorts were performed using protocols described elsewhere29 (link),32 (link),36 (link). Genotyping of the TW2 cases cohorts was performed using the Agena Bioscience MassArray iPLEX platform at the National Center for Genome Medicine, Academia Sinica, Taiwan.
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Candidate SNPs of RIPK1 gene were selected from previous studies, and preliminary analysis of RIPK1 polymorphism was done using the 1000 Genomes database with minor allele frequency (MAF) larger than 0.05 [15 (link)]. Genomic DNA was extracted from peripheral blood samples via a blood DNA kit (GoldMag Co. Ltd., Xi′an, China), and quantified with Nanodrop 2000 (Thermo Scientific, Waltham, Massachusetts, USA). MassARRAY Assay Design 3.0 software was applied for primers design (Table 1). The SNP genotyping was performed using MassARRAY iPLEX platform (Agena Bioscience, San Diego, CA, USA) according to the manufacturer’s instructions [16 (link)]. Finally, the genotyping results were managed and outputted by Agena Bioscience TYPER version 4.0 software.

Primer sequences used for this study

SNPFirst-PCRPSecond-PCRPUEP_DIRUEP_SEQ
rs6907943ACGTTGGATGACCAGGTGTTGGAGTTCAGCACGTTGGATGGGTGTTTGTTTGCAGCTCGTFtgtgTGCAGCTCGTTAGCAT
rs2077681ACGTTGGATGGTGAATTTAACTGCACTGGGACGTTGGATGAACCTCGAGGACATCATGCCRggggtACATCATGCCAAGTGGA
rs9503400ACGTTGGATGAGTAAGTGCTCAGTAAACGGACGTTGGATGTGCTCAAGGCTGTCTAGGTGRggagGGCTGTCTAGGTGTTCTTTG
rs17548629ACGTTGGATGTCAACAGTATCAGCCCTGAGACGTTGGATGTGGCATTCTGGTACCTTCACFccccTCACCCAGCCTGAGTG

SNP Sing nucleotide polymorphism

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This study was divided in two phases: a genomic stage with an AmpliSeq-based discovery approach followed by a validation through iPLEX MassARRAY genotyping of candidate SNPs. In Phase 1 (discovery), we used 40 samples from each cases and controls group for Next Generation Sequencing (NGS)-profiling of selected regions (detailed in the AmpliSeq Library Preparation section below). For this initial phase, the 40 case samples were chosen based on the severity of the disease (determined by the chest X-rays and sputum microscopy). The population characteristics are provided in Table 1. After NGS, association analysis was performed at allele and genotype level. Candidate SNPs were then subjected to a second phase (validation), comprising the genotyping of all remaining cohort samples for the candidate SNPs obtained from the explorative approach. The genotyping was performed using the MassARRAY iPLEX Platform (Agena Bioscience).
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Genomic DNA was extracted from peripheral blood stored with EDTA using blood DNA kit (GoldMag Co. Ltd.). The concentration of the DNA samples was measured with Nanodrop 2000 (Thermo Scientific). In this study, five SNPs in CDKN2B‐AS1 were selected from UCSC database and each candidate SNP had larger than 5% minor allele frequency in Chinese Han population. The primers used in this study were designed using MassARRAY Assay Design 3.0 software (Table S1), and the genotyping was performed on the MassARRAY iPLEX platform (Agena Bioscience) (Sun et al., 2017). We checked the quality of the genotype determination by the same method. We predicted functions of selected polymorphisms by HaployReg v4.1. Agena Bioscience TYPER version 4.0 software was used to perform data management and analyses.
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1,223 germline DNA samples from the validation cohort were genotyped for the KDR Q472H variant using the MassARRAY iPLEX platform (Agena Bioscience, San Diego, CA, USA). The variant was in Hardy-Weinberg equilibrium (p=0.526).
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According to the 1000 Genomes Project (http://www.1000genomes.org/) and dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/) database, five label single nucleotide polymorphisms (SNPs) (rs12138817, rs9440302, rs1198574, rs17371457 and rs7554283) with minor allele frequency (MAF) > 5% were finally selected to evaluate the effect of MIR137HG polymorphisms on LC susceptibility.
Peripheral blood samples from all participants were collected in tubes coated with EDTA and were stored at 80°C. Following the manufacturer’s guidelines, genomic DNA was extracted from participants’ peripheral blood samples using the GoldMag whole blood genomic DNA purification kit (GoldMag Co. Ltd., Xi an, China). DNA concentration was measured with NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).10 (link) The primers for the amplification reactions were designed using the Agena Bioscience Assay Design Suite V2.0 software to perform (Agena Bioscience, San Diego, CA, USA, https://agenacx.com/online-tools/).11 (link) Primers used for this study were listed in Supplementary Table S1. The MassARRAY iPLEX platform and Agena Bioscience TYPER version 4.0 software were used for SNP genotyping and data analysis, respectively.
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