Shrimp alkaline phosphatase

We selected SNPs (rs8051542, rs12443621, rs3803662, rs4784227 and rs3112612) in the TOX3/LOC643714 locus at 16q12.1 from the confirmatory results from GWAS or meta-analysis in multiple populations [6 (link),7 (link),11 (link),20 (link)].
The five SNPs were genotyped using the SEQUENOM MassARRAY matrix-assisted laser desorption ionization-time of flight mass spectrometry platform (Sequenom, San Diego, CA, USA). Primers for multiplex PCR and extended reactions were designed using proprietary software (Assay Designer, version 3.1) provided by Sequenom Inc (San Diego, CA, USA). In accordance with the manufacturer’s instructions, SNPs were genotyped using Sequenom MassARRAY genotyping technology (Sequenom, San Diego, CA, USA) and amplified in multiplex PCR by a standard PCR protocol. The genomic amplification product was cleaned using shrimp alkaline phosphatase (Sequenom, San Diego, CA, USA) to neutralize any unincorporated dNTPs, followed by a single-base extension reaction using the iPLEX enzyme (Sequenom, San Diego, CA, USA) and mass-modified terminators (Sequenom, San Diego, CA, USA). The products of the iPLEX reaction were desalted and transferred onto a SpectroCHIP (Sequenom, San Diego, CA, USA) by the MassARRAY nanodispenser (Sequenom, San Diego, CA, USA), which was then analyzed by the MassARRAY analyzer by combining base calling with the clustering algorithm.

EpiTYPER assays were designed for a total of 169 CpG sites spread over 5 CGIs using the EpiDesigner software (Sequenom, San Diego, CA, USA; EpiDesigner.com" xmlns:xlink="http://www.w3.org/1999/xlink">www.EpiDesigner.com). After PCR amplification of bisulfite-converted DNA, the amplicons were treated with Shrimp Alkaline Phosphatase (Sequenom) followed by in vitro transcription. The resulting product was treated with RNase A to cleave the RNA at each U nucleotide using the T-Cleavage MassCLEAVE Kit (Sequenom). The fragments were then conditioned with Clean Resin (Sequenom) with the protocol provided in the kit and dispensed onto a SpectroCHIPII (Sequenom) and then analyzed on a MassARRAY Workstation in EpiTYPER mode. Quality control and initial data analysis were performed with the use of EpiTYPER (version 1.2, Sequenom). Oligonucleotide sequences for the EpiTYPER assays are provided in Supplementary Table 2.

We designed the primers using Assay Design 3.1 software (Sequenom Inc., San Diego, CA, USA), whose details were presented in Supplementary Table S1.
All consenting participants contributed a 2-mL saliva sample using DNA saliva self-collection tube (Beijing Thinkout Sci-Tech Co., Ltd., Beijing, China). DNA was extracted and purified from all saliva samples via a DNA purification kit (Beijing BioTeke Co., Ltd., Beijing, China). DNA fragments were amplified by the polymerase chain reaction (PCR), whose reaction volume was 5 μL containing 20 ng of genomic DNA template. The PCR product was digested with shrimp alkaline phosphatase (Sequenom Inc., San Diego, CA, USA). Following digestion, the amplified fragment was extended in a 9 μL reaction volume and resolved on a 4% agarose gel. All SNPs were genotyped using the high-throughput genotyping platform, conducted by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and the data was collected via the TYPER 4.0 in the MassARRAY System (Sequenom Inc., San Diego, CA, USA). Furthermore, genotyping was repeated in 5% of all samples to verify the accuracy of the results, revealing that the proportion of concordance was greater than 99%.

Unincorporated dNTPs in the amplification products were dephosphorylated by adding 1.7 μL Dnase free water and 0.3 μL (0.5 U) shrimp alkaline phosphatase (SAP) (Sequenom, Inc., San Diego, CA, USA). Each reaction was incubated at 37 °C for 40 min, and SAP was then heat inactivated for 5 min at 85 °C. Subsequently, samples were incubated for 3 h at 37 °C with 5 μL of T‐cleavage reaction mix (Sequenom), containing 3.21 μL RNAse‐free water, 0.89 μL 5× T7 polymerase buffer, 0.22 μL T‐cleavage mix, 0.22 μL 100 mm DTT, 0.40 μL T7 RNA & DNA polymerase and 0.06 μL RNAse A, for concurrent in vitro transcription and base‐specific cleavage. The samples of cleaved fragments were then diluted with 20 μL water. Conditioning of the cleavage reaction was carried out by adding 6 mg of clean resin.