Massarray analyzer 4

Manufactured by Agena

Sourced in United States

The MassARRAY Analyzer 4 is a high-throughput mass spectrometry system used for genetic analysis. It is designed to perform rapid and accurate detection and quantification of DNA and RNA molecules.

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Lab products found in correlation

Spectrochip 2, by Agena (2 mentions) Spectrochip array, by Agena (2 mentions) Nanodispenser rs1000, by Agena (2 mentions) Massarray system, by Agena (2 mentions) Taqman validated snp genotyping assay kits, by Thermo Fisher Scientific (1 mentions) Genemapper v3, by Thermo Fisher Scientific (1 mentions) Iplex gold, by Agena (1 mentions) Typer 4, by Agena (1 mentions) Iplex gold assay, by Labcorp (1 mentions) Iplex gold assay, by Agena (1 mentions) Massarray, by Agena (1 mentions) Hot firepol dna polymerase, by Solis BioDyne (1 mentions) Typer software version 4, by Agena (1 mentions) Shrimp alkaline phosphatase, by Agena (1 mentions) Nanodrop 8000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions) Vacutainer tube, by BD (1 mentions) Qiaamp dna blood mini kit, by Qiagen (1 mentions)

14 protocols using massarray analyzer 4

Genotyping ANKK1 rs2734849 by MassARRAY

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A standard phenol–chloroform micro method (as described by Ivanova et al., 2012) was used to isolate DNA from the leukocytes in the whole peripheral blood samples after pre‐freezing of the blood.
Genotyping for ANKK1 rs2734849 was carried out on the MassARRAY® Analyzer 4 (Agena Bioscience™) using the set SEQUENOM Consumables iPLEX Gold 384. DNA sample preparation for SEQUENOM MassARRAY® Analyzer 4 includes several steps: a standard PCR reaction to obtain the amplification products, a shrimp alkaline phosphatase reaction to neutralize the unincorporated dNTPs in the amplification products, the PCR iPLEX Gold extension reaction, and then placing the samples on a special chip (SpectroCHIP Array) using NanoDispenser RS1000 prior to loading them into the analyzer.

Fedorenko O.Y., Paderina D.Z., Loonen A.J., Pozhidaev I.V., Boiko A.S., Kornetova E.G., Bokhan N.A., Wilffert B, & Ivanova S.A. (2020). Association of ANKK1 polymorphism with antipsychotic‐induced hyperprolactinemia. Human Psychopharmacology, 35(4), e2737.

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FTO Genetic Variants Identification

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Genotyping of six single nucleotide polymorphisms of FTO gene (rs7185735, rs9939609, rs1421085, rs1861868, rs3751812, and rs8050136) was carried out using the mass spectrometer MassARRAY^® Analyzer 4 (Agena Bioscience™) and a QuantStudio ™ 3D Digital PCR System Life Technologies (Applied Biosystems) amplifier using TaqMan Validated SNP Genotyping Assay kits (Applied Biosystems), on the base The Core Facility “Medical Genomics”, Tomsk NRMC. The criteria for selecting the variants mentioned were a) their citation in the relevant scientific literature as described in the introduction and b) a minor allele frequency (MAF) of at least 5%. Basic information of these SNPs is described in

Supplementary Table 1

Boiko A.S., Pozhidaev I.V., Paderina D.Z., Bocharova A.V., Mednova I.A., Fedorenko O.Y., Kornetova E.G., Loonen A.J., Semke A.V., Bokhan N.A, & Ivanova S.A. (2021). Search for Possible Associations of FTO Gene Polymorphic Variants with Metabolic Syndrome, Obesity and Body Mass Index in Schizophrenia Patients. Pharmacogenomics and Personalized Medicine, 14, 1123-1131.

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Genotyping SNPs Using MassARRAY

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SNPs were analyzed on the MassARRAY Analyzer 4 system using iPLEX Gold chemistry (both Agena Biosciences, San Diego, CA, USA) using the primers in Supplementary Table S11 and the recommended manufacturer’s protocol. Genotypes were called using Typer 4.0 (Agena Biosciences). The AVPR1A RS3 polymorphism primers and methods are also listed in Table S11. Primers were adapted from that used by Tansey et al. to match the Human Feb. 2009 (GRCh37/hg19) assembly and to elevate the Tm but otherwise maintain the amplified position⁷³. Results were analyzed using the software GeneMapper v. 3.7 (Life Technologies).

Baribeau D.A., Dupuis A., Paton T.A., Scherer S.W., Schachar R.J., Arnold P.D., Szatmari P., Nicolson R., Georgiades S., Crosbie J., Brian J., Iaboni A., Lerch J, & Anagnostou E. (2017). Oxytocin Receptor Polymorphisms are Differentially Associated with Social Abilities across Neurodevelopmental Disorders. Scientific Reports, 7, 11618.

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Genotyping Five Thrombosis-Associated SNPs

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We genotyped the SNPs rs8176719 in ABO (ABO blood group), rs6025 in F5 (FVL), rs1799963 in F2 (prothrombin G20210A), and rs2036914 in F11 using the Sequenom platform, and rs2066865 in FGG using the TaqMan platform, as previously described.22 For Sequenom, samples were genotyped using the Sequenom iPlex Gold Assay according to the recommended protocol, using an initial input of 10 to 20 ng DNA, and were analyzed using the MassARRAY Analyzer 4 (Agena Bioscience, San Diego, CA, USA). For TaqMan, an initial input of 100 ng of DNA was used, and samples were genotyped using the 7900HT (Applied Biosystems, Foster City, CA, USA) according to the recommended protocol.
Subjects were categorized as carriers of the prothrombotic risk gene when ≥1 risk allele was present. For rs2036914, the minor allele was associated with lower risk of VTE, and therefore we considered the major allele as the risk allele. Based on the paper by de Haan et al,5 we composed a 5‐SNP score by summarizing the number of risk alleles from the 5 sequenced SNPs. These were further categorized into 0 to 1, 2, 3, and ≥4 risk alleles.

Sejrup J.K., Morelli V.M., Løchen M., Njølstad I., Mathiesen E.B., Wilsgaard T., Hansen J, & Brækkan S.K. (2020). Myocardial infarction, prothrombotic genotypes, and venous thrombosis risk: The Tromsø Study. Research and Practice in Thrombosis and Haemostasis, 4(2), 247-254.

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Genotyping of NUDT15 Variants

Cited 1 time

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Genotyping of three variant NUDT15 alleles [*2 (*3) (rs116855232), *4 (rs147390019) and *5 (rs186364861)] was performed using a comprehensive pharmacogenetic genotyping panel (S. A. Scott et al., 2020 (link)), which employs multiplex PCR and single base extension (SBE) using the Agena^® SpectroCHIP^® II and MassARRAY^® Analyzer 4 platform as per manufacturer instructions (Agena Biosciences, San Diego, CA). Genotypes at the targeted loci were determined by SBE peak intensity and Typer software v4.1 (Agena Biosciences), and NUDT15 diplotypes were inferred by a haplotype translation table and Typer software v4.1. Of note, the Agena chemistry was unable to interrogate the c.50_55dup (rs746071566) variant, which independently defines *6 and is found in cis with c.415C>T (rs116855232) on the *2 haplotype. Given that c.415C>T (rs116855232) is found on both *2 and *3, the Agena genotyping panel is unable to distinguish between NUDT15*2 and *3 when c.415C>T (rs116855232) is detected (S. A. Scott et al., 2020 (link)).

Scott E.R., Yang Y., Botton M.R., Seki Y., Hoshitsuki K., Harting J., Baybayan P., Cody N., Nicoletti P., Moriyama T., Chakraborty S., Yang J.J., Edelmann L., Schadt E.E., Korlach J, & Scott S.A. (2022). Long-read HiFi sequencing of NUDT15: Phased full-gene haplotyping and pharmacogenomic allele discovery. Human mutation, 43(11), 1557-1566.

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Genotyping of Candidate SNPs

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Candidate SNPs from the exploration cohort, as well as a 7 wild-card SNPs not included on the Cardio-Metabo chip, were genotyped using the Agena Biosciences MassARRAY ® platform (formerly Sequenom). SNP multiplexes were designed using Assay Design Suite v.1.0 software (Agena Bioscience, San Diego, CA, US). Genotyping was performed according to the manufacturer's protocol using IPLEX Gold assay (Agena Bioscience, San Diego, CA, US) and analyzed using the MassARRAY Analyzer 4 platform. Mass signals for the different alleles were captured with high accuracy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Genotype clustering and individual sample genotype calls were generated using Sequenom TyperAnalyzer v.4.0 software (Agena Bioscience, CA, US).

Velle-Forbord T., Eidlaug M., Debik J., Sæther J.C., Follestad T., Nauman J., Gigante B., Røsjø H., Omland T., Langaas M, & Bye A. (2019). Circulating microRNAs as predictive biomarkers of myocardial infarction: Evidence from the HUNT study. Atherosclerosis, 289.

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Methylation Analysis of GPR15 in Smokers

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We also compared methylation patterns between the smokers and never-smokers using MassArray EpiTyper DNA methylation technology (Agena Bioscience, San Diego, CA). Samples were prepared using an EpiTyper T Complete Reagent Set according to the manufacturer's instructions (Agena Bioscience). The bisulfite-treated DNA (25 ng) was amplified with Hot FirePol DNA Polymerase (Solis BioDyne, Tartu, Estonia), and CpG methylation was determined by the MassArray Analyzer 4 (Agena Bioscience). The specific primers were designed with the EpiDesigner software beta version (Agena Bioscience), and the primer sequences for GPR15 were 5 0 -AGGAAGAGAGTATTGTTTTTTTGGGTGGATAAAGA-3 0 and 5 0 -CAGTAATACGACTCACTATAGGGAGAAGG-CTCAATAACAAATCACAATACTCAACAAAA-3 0 .

Kõks G., Uudelepp M.L., Limbach M., Peterson P., Reimann E, & Kõks S. (2015). Smoking-induced expression of the GPR15 gene indicates its potential role in chronic inflammatory pathologies. The American journal of pathology, 185(11).

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Comprehensive Pharmacogenetic Panel Analysis

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The comprehensive pharmacogenetic panel uses multiplex polymerase chain reaction (PCR) and single base extension (SBE) using the Agena SpectroCHIP II and MassARRAY Analyzer 4 platform, as per manufacturer instructions (Agena Biosciences, San Diego, CA). In brief, for each sample 10–20 ng of genomic DNA was amplified in six independent 5 µl multiplex PCR reactions, which consisted of an initial denaturation step at 95°C for 2 minutes followed by 45 cycles (95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes). Amplicons were inactivated by shrimp alkaline phosphatase (Agena Biosciences, San Diego, CA) and subjected to six corresponding multiplex SBE reactions using 2 µl of SBE reagent (Agena Biosciences), which consisted of an initial denaturation step at 95°C for 30 seconds followed by 40 cycles (95°C for 5 seconds (52°C for 5 seconds and 80°C for 5 seconds) × 5). SBE products were conditioned with resin to remove salts, spotted on a SpectroCHIP II array, and read on the MassARRAY Analyzer 4 system. Genotypes at all targeted loci were determined by SBE peak intensity and Typer software version 4.1 (Agena Biosciences, San Diego, CA), and diplotypes for selected genes were inferred by a haplotype translation table and Typer software version 4.1.

Scott S.A., Scott E.R., Seki Y., Chen A.J., Wallsten R., Owusu Obeng A., Botton M.R., Cody N., Shi H., Zhao G., Brake P., Nicoletti P., Yang Y., Delio M., Shi L., Kornreich R., Schadt E.E, & Edelmann L. (2020). Development and Analytical Validation of a 29 Gene Clinical Pharmacogenetic Genotyping Panel: Multi‐Ethnic Allele and Copy Number Variant Detection. Clinical and Translational Science, 14(1), 204-213.

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UltraSEEK Mutation Detection Assay

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UltraSEEK assays were designed using the AgenaCx online assay design software which automatically selects the PCR and extension primers (Supplementary Table 4), and adds to each reaction control assays for PCR and capturing. All oligonucleotides were obtained from Integrated DNA Technologies and control oligos from Agena Bioscience GmbH. Reactions were performed as described before^{36 (link)}, using reagents obtained from Agena Bioscience. Briefly, PCR (45 cycles) was followed by shrimp alkaline phosphatase treatment and single base primer extension using biotinylated ddNTPs specific for the mutant alleles. After capture of the extended primers using streptavidin-coated magnetic beads, a cation-exchange resin was added for cleaning and 10-15 nl of the reaction was transferred to a SpectroCHIP® Array (a silicon chip with pre-spotted matrix crystals) using an RS1000 Nanodispenser (Agena Bioscience). Data were acquired via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a MassARRAY Analyzer 4 (Agena Bioscience). After data processing, a spectrum was produced with relative intensity on the y-axis and mass/charge on the x-axis. Typer Analyzer software was used for data analysis and report generation.

Weerts M.J., Timmermans E.C., Vossen R.H., van Strijp D., Van den Hout–van Vroonhoven M.C., van IJcken W.F., van der Zaag P.J., Anvar S.Y., Sleijfer S, & Martens J.W. (2018). Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue. Scientific Reports, 8, 2261.

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Ultrasensitive Mutational Analysis of Melanoma

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Mutational analysis was performed using the UltraSEEK™ Melanoma Panel v1.0 (Agena Bioscience, Hamburg, Germany), interrogating 61 clinically relevant variants across 13 genes, including BRAF, NRAS, KIT and MAP2K1, detected at as low as 0.1% minor allele frequency. Reactions were performed as described before [46 (link)]. In brief, PCR (45 cycles) was followed by shrimp alkaline phosphatase treatment and single base primer extension, using biotinylated ddNTPs specific for the mutant alleles. After capture of the extended primers using streptavidin-coated magnetic beads, a cation-exchange resin was added for cleaning, and 10–15 nL of the reaction was transferred to a SpectroCHIP^® Array (a silicon chip with pre-spotted matrix crystals) using an RS1000 Nanodispenser (Agena Bioscience). Data were acquired via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, using a MassARRAY Analyzer 4 (Agena Bioscience, Hamburg, Germany). After data processing, a spectrum was produced with relative intensity on the y-axis and mass/charge on the x-axis. Typer Analyzer software was used for data analysis and automated report generation. Sanger sequencing was performed to verify mutations detected by the UltraSEEK™ Melanoma Panel, and only mutations which were detected in both assays (98%) were used for further analysis.

Gorges K., Wiltfang L., Gorges T.M., Sartori A., Hildebrandt L., Keller L., Volkmer B., Peine S., Babayan A., Moll I., Schneider S.W., Twarock S., Mohr P., Fischer J.W, & Pantel K. (2019). Intra-Patient Heterogeneity of Circulating Tumor Cells and Circulating Tumor DNA in Blood of Melanoma Patients. Cancers, 11(11), 1685.

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