Genomic DNA from white blood cells of probands and their family members was used for genotyping MLH1 (NM_000249.3), APEX1 (NM_001641.3), MUTYH (NM_012222.2), OGG1 (NM_016828.2), NUDT1 (NM_002452.3), XRCC5 (NM_021141.3), XPA (NM_000380.3), and ERCC2 (NM_000400.3) (Table 1 ). Genotyping was performed using Sequenom iPLEX MassARRAY (Sequenom, Inc., San Diego, CA, USA). Matrix‐assisted laser desorption ionization time of flight (MALDI‐TOF) spectroscopy was performed using the Sequenom MassARRAY platform and iPLEX GOLD chemistry as described in our previous studies (Kamiza et al., 2015 , 2016 , 2018 ). We added 10 ng of template DNA in polymerase chain reaction (PCR) mixture containing Qiagen HotStarTaq. We conducted primer extensions and shrimp alkaline phosphatase by using guidelines from Sequenom. Primers used for PCR were from Integrated DNA Technologies (OH, USA). Assays were designed using MassARRAY Assay Design, Version 3.1 (Sequenom). We repeated 10% of randomly selected samples for quality control and results showed 100% concordance for all the SNPs.
Sequenom massarray
Manufactured by Labcorp
Sourced in United States
The Sequenom MassARRAY is a sensitive and accurate genetic analysis system used for high-throughput genotyping and targeted next-generation sequencing. It utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry technology to determine nucleic acid sequences and detect genetic variations.
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Lab products found in correlation
Iplex gold genotyping assay, by Labcorp (4 mentions)
Massarray designer, by Labcorp (4 mentions)
Qiamp dna blood mini kit, by Qiagen (2 mentions)
Iplex gold genotyping asssy, by Labcorp (2 mentions)
Abi 7900ht, by Thermo Fisher Scientific (2 mentions)
Massarray assay design version 3, by Labcorp (1 mentions)
Hotstartaq, by Qiagen (1 mentions)
Sequenom snp assay design software version 3, by Labcorp (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Abi 7300 real time polymerase chain reaction system, by Thermo Fisher Scientific (1 mentions)
Iplex assay, by Labcorp (1 mentions)
Massarray software, by Labcorp (1 mentions)
Massarray assay design 4, by Labcorp (1 mentions)
Massarray typer 4, by Labcorp (1 mentions)
Faststart taq dna polymerase, by Roche (1 mentions)
Software, by MacVector (1 mentions)
Human omni express exome bead chip kit, by Illumina (1 mentions)
13 protocols using sequenom massarray
1
Genotyping Genetic Variants in Proband Families
2
Genotyping of CCL17 and 25 SNPs
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Peripheral blood of the three experimental groups including BD and VKH disease patients and the controls was subjected to genomic DNA extraction with the QIAmp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA, USA) and the DNA was stored at −80°C. The Applied Biosystems 7500 Real-Time PCR system was utilized to genotype CCL17/rs223828 (TagMan assay ID: C_30530263_10) by the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA). Genotype identification of the other 25 SNPs was conducted with the iPLEX Gold genotyping assay and Sequenom MassARRAY (Sequenom, CA, USA). Sequenom SNP Assay Design software version 3.0 was used to design primers of iPLEX reactions. Primer sequences used were shown in Table 1 . The protocol and experimental requirements were performed strictly based on the instructions.
3
Genetic Variant Profiling of XAB2 in Chinese
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Based on the Han Chinese in Beijing (CHB) population data from HapMap database, we used Haploview 4.2 program to select candidate tag SNPs with an r2 threshold of 0.80 and minor allele frequency (MAF) greater than 1 %. For XAB2 gene, we extended the 5′- and 3′-untranslated regions (UTR) to include the 5′-UTR and 3′-UTR most SNP. As a result, 5 tagSNPs (2 in 5′ UTR, 2 in intron, 1 in exon region) in XAB2 were included, which represent the common genetic variants in Chinese population. Genotyping was performed at Bomiao Tech (Beijing, China) using iPlex Gold Genotyping Asssy and Sequenom MassArray (Sequenom, San Diego, CA, USA). Sequenom’s MassArray Designer was used to design PCR and extension primers for each SNP. The information on assay conditions and the primers are available upon request. Genotyping quality control consisted of no-temple control samples for allele peaks and verifying consistencies in genotype calls of 2 % randomly selected duplicate sample. In addition, we excluded individuals and SNPs based on genotyping quality (<90 % call rate).
4
Genotyping Ivermectin Response Worms
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A subset of genome-wide SNPs (160 in total) were chosen to explore ivermectin association and population structure further by Sequenom genotyping of individual adult female worms phenotyped for ivermectin response. DNA from 592 individual female worms (described in Table G in S1 Text ) was prepared for Sequenom MassARRAY genotyping (Sequenom, Inc., San Diego, CA, USA)[78 (link)]. Due to the DNA quantity requirements for Sequenom analysis (600 ng per sample), many individual worm DNA samples (401 of 436 samples from Cameroon, 96 of 156 samples from Ghana) required whole genome amplification to increase the DNA concentration. This was performed using the REPLI-g screening kit (Qiagen, Toronto, ON, Canada). DNA concentrations of all samples were quantified using Quant-iT Pico Green dsDNA Assay Kit (Life Technologies Inc, ON, Canada), before sending to the McGill University and Génome Québec Innovation Centre for genotyping.
5
Genotyping of Inflammatory SNPs
6
Genotyping of Common Chinese Variants
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Based on the Chinese population data from HapMap database, we used HaploView 4.2 program to select the candidate tag SNPs with an r2 threshold of 0.8 and minor allele frequency (MAF) greater than 1%. Under this criteria, totally 11 tag SNPs were selected. Additionally, we added two potential functional SNPs, rs9429942 and rs6691117 18 (link), 19 (link). Therefore, we included 13 SNPs in our study, which represents common genetic variants in Chinese population.
Genotyping was performed at Bomiao Tech (Beijing, China) using iPlex Gold Genotyping Assay and Sequenom MassArray (Sequenom, San Diego, CA, USA). Sequenom's MassArray Designer was used to design PCR and extension primers for each SNP. The PCR primers used are available upon request. Genotyping quality control consisted of no-temple control samples for allele peaks and verifying consistencies in genotype calls of 2% randomly selected duplicate samples. In addition, two control samples were included on each plate as genotyping controls for inter-plate reproducibility. Hardy-Weinberg Equilibrium (HWE) was also evaluated in unrelated controls.
Genotyping was performed at Bomiao Tech (Beijing, China) using iPlex Gold Genotyping Assay and Sequenom MassArray (Sequenom, San Diego, CA, USA). Sequenom's MassArray Designer was used to design PCR and extension primers for each SNP. The PCR primers used are available upon request. Genotyping quality control consisted of no-temple control samples for allele peaks and verifying consistencies in genotype calls of 2% randomly selected duplicate samples. In addition, two control samples were included on each plate as genotyping controls for inter-plate reproducibility. Hardy-Weinberg Equilibrium (HWE) was also evaluated in unrelated controls.
7
Multiplex SNP Genotyping Using MassARRAY
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The assay was designed using MassARRAY® software (version 4.0; Sequenom, San Diego, CA, USA). The 138 mutations were assigned to six multiplex assays. PCR primers were designed using Mass ARRAY® Assay Design 4.0 Software (Sequenom) (Table II ). PCR was first performed using the following protocol: 4 min at 95°C for activation of Faststart taq DNA polymerase (Roche Diagnostics, Basel, Switzerland, cat. no. 12032937001) and 30 sec at 95°C, 30 sec at 56°C, 1 min at 72°C for 45 cycles, followed by 5 min at 72°C. The PCR products were subjected to shrimp alkaline phosphatase (SAP) reaction for the degradation of residual dNTPs. The SAP reaction was performed as follows: 40 min at 37°C and 5 min at 85°C. Following this, extension reaction was performed by the following protocol: 30 sec at 94°C, 40 cycles for 5 sec at 94°C, from 5 sec at 52°C to 5 sec at 80°C for 5 cycles, and finally 3 min at 72°C. Then, the products were desalted using resin. The final products were analyzed by MALDI-TOF mass spectrometry (Mass ARRAY® Typer 4.0.5 Software, Sequenom) to identify the mass. SNP genotyping was performed on SEQUENOM®MassARRAY® platform using the iPLEX™ assay (Sequenom) (37 (link)).
8
Genotyping of Common Variants in Chinese Population
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Based on the Chinese population data from HapMap database, we used Haploview 4.2 program to select candidate tag SNPs with an r2 threshold of 0.80 and minor allele frequency (MAF) greater than 1%. Furthermore, we also added two potential functional polymorphisms, rs9429942 and rs6691117 [42 (link),43 (link)]. Therefore, we included 13 SNPs in our study, which represents common genetic variants in Chinese population.
Genotyping was performed at Bomiao Tech (Beijing, China) using iPlex Gold Genotyping Asssy and Sequenom MassArray (Sequenom, San Diego, CA, USA). Sequenom’s MassArray Designer was used to design PCR and extension primers for each SNP. Primer information for selected tag SNPs was listed in Table5 .
Genotyping was performed at Bomiao Tech (Beijing, China) using iPlex Gold Genotyping Asssy and Sequenom MassArray (Sequenom, San Diego, CA, USA). Sequenom’s MassArray Designer was used to design PCR and extension primers for each SNP. Primer information for selected tag SNPs was listed in Table
9
TXNRD1 Genetic Variation Protocol
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14 SNPs covering the whole TXNRD1 genetic variability were prioritized by a tagging approach, attempting to choose those most likely to be of functional relevance (nonsynonymous SNPs, SNPs located in the 5′ and 3′ UTR regions). SNPs associated with health status and longevity in Northern Europeans [14 (link), 15 (link)] were also chosen. SNPs with a minor allele frequency (MAF) less than 5% were excluded from the analysis.
Multiplex SNP genotyping was performed using iPLEX Gold Genotyping Assay and Sequenom MassARRAY (Sequenom, San Diego, CA, USA) according to manufacturer's instructions. Sequenom's MassARRAY Designer was used to design PCR and extension primers for each of the 14 SNPs selected. However, four of them (rs10861169, rs10861197, rs10047589, and rs4964287) were skipped by the software for primers design and were not analyzed in this paper. The details for genotyped SNPs are listed inTable 1 .
Multiplex SNP genotyping was performed using iPLEX Gold Genotyping Assay and Sequenom MassARRAY (Sequenom, San Diego, CA, USA) according to manufacturer's instructions. Sequenom's MassARRAY Designer was used to design PCR and extension primers for each of the 14 SNPs selected. However, four of them (rs10861169, rs10861197, rs10047589, and rs4964287) were skipped by the software for primers design and were not analyzed in this paper. The details for genotyped SNPs are listed in
10
Genotyping Protocols for Genetic Studies
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In the MDCS cohort, a MALDI-TOF mass spectrometer (Sequenom MassArray, Sequenom, San Diego, CA, USA) was used to genotype DNA samples using Sequenom reagents and protocols. In the case where a candidate SNP failed the genotyping, a “proxy SNP” was used in its place. Proxy SNPs were identified using SNAP version 2.2.2 when commercial primers were not available. SNPs that failed Sequenom genotyping were alternatively genotyped individually using TaqMan, KASPar allelic discrimination on an ABI 7900HT (Applied Biosystems, Life Technologies, Carlsbad, CA, USA), per manufacturer’s instructions. In the MPP, blood samples were taken, on average, 25 years after study baseline, and was thus excluded from the MR analysis to avoid collider bias [24 (link), 25 (link)]. In the UK-biobank, Affymetrix (ThermoFisher Scientifics) performed genotype calling on two closely related, but custom-designed arrays. Approximately 50,000 participants were ran on UK BiLEVE Axiom array and the remaining 450,000 were ran on UK-Biobank Axiom array. A detailed description of the genotype process and internal quality control is described elsewhere [21 (link)].
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