Iplex assay

The g.71874104G>A and g.71833755T>C loci in the HIRA gene were selected for genotyping in 760 samples from Small Tail Han, Tan, Sunite, Suffolk, Dorper, and Prairie Tibetan sheep. Genotyping was performed using PCR and primer extension and mass spectrometric analysis (iPlex assay, Sequenom, San Diego, CA, USA) on a Sequenom MassArray according to the manufacturer’s instructions (http://www.sequenom.com). Polymerase chain reactions were carried out in 5 µL containing 1.0 µL 20–50 ng/µL genomic DNA, 0.5 µL 10 × PCR buffer, 0.4 µL 25 mmol/L MgCl₂, 0.1 µL 25 µmol/L dNTP, 1.0 µL PCR Primer mix, 0.2 µL Taq DNA polymerase (Promega, Madison, WI, USA), and ddH₂O. PCR conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 60 s, with a final extension at 72 °C for 10 min. Primer extension reactions were carried out in 2 µL containing 0.2 µL iplex Buffer, 0.2 µL Terminator mix, 0.94 µL Extend primer mix, 0.04 µL iplex Enzyme, and ddH₂O. Extension conditions were as follows: initial denaturation at 94 °C for 30 s, followed by 40 cycles of denaturation at 94 °C for 5 s, annealing at 52 °C for 5 s, with a final extension at 72 °C for 3 min. Only those samples with a >95% success rate and only those SNPs with a genotype success rate of >95% were included in the analysis.

Genomic DNA was extracted from EDTA blood or mouthwash samples using the FlexiGene DNA kit (Qiagen GmbH, Hilden, Germany) and quantified using Quant‐iT Pico Green dsDNA reagent kit (Invitrogen/Life Technologies, Darmstadt, Germany). Of 492 selected SNPs, 447 passed quality control after genotyping (for 45, success rate was below 95%, seven were not in Hardy–Weinberg equilibrium (HWE) and were selected to be genotyped on the customized GoldenGate assay (Illumina, San Diego, CA) 23. The iPLEX assay (Sequenom, Hamburg, Germany) for the MassArray system was used to genotype five SNPs that failed genotyping on the Illumina GoldenGate platform 24. Quality of genotyping was high with concordance of duplicates from Centre d'Etude du Polymorphisme Humain (Paris, France) and control samples above 98%. The two selected non‐SNP polymorphisms in the TYMS gene were genotyped using fragment analysis and single‐strand conformation polymorphism in the laboratory of Dr. Ulrich at the National Center for Tumor Diseases in Heidelberg, Germany.

We collected the patients’ paraffin sections after esophagectomy from the Department of Pathology, Tongji Hospital. Genomic DNA was extracted with QIAamp DNA FFPE tissue kits (56404; Qiagen, Dusseldorf, Germany) from paraffin sections. Haploview software was used to choose the single nucleotide polymorphisms (SNPs) in the p14ARF/MDM2/TP53 pathway. We chose three key genes (p14ARF, MDM2 and TP53) along the p14ARF/MDM2/TP53 pathway by a candidate gene approach [20 (link)]. All of the SNPs had minor allele frequencies of greater than 20% in the Chinese population based on the HapMap HCB data, and all correlated alleles were captured at r² > 0.8. We only selected SNPs that were located in the 5′ or 3′-UTR gene region, or were found to be associated with the risk of ESCC by our previous reports, for example rs1042522. SNPs in strong linkage disequilibrium with selected SNPs or SNPs that cannot be determined in a well were excluded. Six SNPs were finally selected. For all six SNPs, genotypes were determined using the MassArray system (Sequenom iPLEX assay, San Diego, CA). The sample DNA was amplified by a multiplex PCR reaction, and the PCR products were then used for a locus-specific single-base extension reaction. Finally, the resulting products were desalted and transferred to a 384-element SpectroCHIP array. The alleles were discriminated by mass spectrometry (Sequenom).

Except for rs1333040, individual samples were genotyped using Sequenom's (San Diego, USA) iPlex assay (primer extension of multiplex products with detection by matrix assisted laser desorption/ionization time-of-flight mass spectrometry) following manufacturer's protocol and detected in a Sequenom MassArray K2 platform. The primer sequences (S1 Table) were designed using Sequenom's MassARRAY Assay Design 3.0 software. Genotyping was performed in the Genomics Unit of the Instituto Gulbenkian de Ciência.
For rs1333040, individuals were genotyped using the TaqMan SNP Genotyping Assay-on-demand C_8766795_10 (Life Technologies, USA), with the TaqMan Genotyping Master Mix on a ViiA 7 Real-Time PCR System.
Extensive quality control was performed using eight HapMap (http://hapmap.ncbi.nlm.nih.gov/) controls of diverse ethnic affiliation, sample duplication within and across plates, Mendelian inheritance check in one large pedigree (without IA), Hardy-Weinberg equilibrium (HWE) in the control group (P>0.001), and a minimum of 90% sample and 85% SNP call rates. Genotype determinations were performed blinded to affection status.