DNA obtained from saliva samples (OG-100; OG-25; Oragene; DNA Genotek) was purified, extracted, and hydrated; it was stored at −80°C when not in use. Primers were designed using Spectro DESIGNER software (Sequenom). Following a PCR, an iPLEX mass EXTEND reaction was performed. After baseline correction and peak identification, Sequenom SPECTROTYPER software was used to analyze resulting spectra. Concordance for duplicate DNA in the current sample was 100%. COMT rs4680 did not deviate from Hardy–Weinberg equilibrium (HWE; all ethnicities: χ2=0.372, p=.54; European American sample only: χ2=1.20, p=.27; sample 1: χ2=.055, p=.82; sample 2: χ2=1.388, p=.24).
Spectrotyper software
Manufactured by Labcorp
The SpectroTYPER software is a laboratory analysis tool that is used to interpret and analyze spectroscopic data. It provides a platform for visualizing and processing data from various spectroscopic techniques, such as UV-Vis, FTIR, and Raman spectroscopy.
Automatically generated - may contain errors
Lab products found in correlation
Massarray system, by Labcorp (3 mentions)
Massarray platform, by Labcorp (2 mentions)
Spectrodesigner software, by Labcorp (1 mentions)
Massarray technology, by Labcorp (1 mentions)
Massarray iplex system, by Labcorp (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Massarray mass spectrometer, by Labcorp (1 mentions)
Massarray assay design 3, by Labcorp (1 mentions)
Autoflex maldi tof mass spectrometer, by Labcorp (1 mentions)
Iplex enzyme, by Labcorp (1 mentions)
Ezna tm blood dna midi kit, by Omega Bio-Tek (1 mentions)
Spectrochip, by Labcorp (1 mentions)
Abi geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
9 protocols using spectrotyper software
1
Catechol-O-Methyltransferase Genotyping
2
Genotyping Using Sequenom Mass Array
3
Genotyping Genetic Variants using Sequenom iPLEX
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All selected genetic variants were genotyped using Sequenom iPLEX MassARRAY system (Sequenom, San Diego, CA) [40 ] at the Australian Genome Research Facility, Australia. The workflow of the Sequenom iPLEX reaction included locus specific polymerase chain reaction, locus specific primer extension reaction and annealing of oligonucleotide primer at the upstream of the polymorphic site being genotyped. Then the primer and amplified target DNA were incubated with mass modified dideoxynucleotide terminators. The primer extension was made according to the sequence of the variant site. Mass of the extended primer was determined through the use of matrix-assisted laser desorption ionization time of flight mass spectrometry. The alleles present at the polymorphic site of interest were assessed based on the sequence specified by the mass of the primer. The mass of the observed primers were translated into a genotype by the SpectroTYPER software of the Sequenom system [40 ].
4
Genotyping of Gemcitabine Transport Genes
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Genomic DNA was extracted from peripheral blood leukocytes using the Wizard Genomic DNA Purification Kit according to the manufacturer’s instructions (Promega, Madison, WI). The 103 genetic polymorphisms of the 23 genes involved in gemcitabine transport and metabolism were selected for genotyping based on a previous study.10 (link) The candidate genetic polymorphisms were genotyped using the MassARRAY system (Sequenom, Inc., San Diego, CA) and Birdseed calling algorithm in SpectroTYPER software (Sequenom, Inc.). The 28-bp tandem repeat in the TYMS 5ʹ-untranslated enhanced region (TSER) and a 6-bp deletion/insertion in the TYMS 3ʹ-untranslated region (TS 3′-UTR) was detected using a protocol previously described.18 (link) Sequencing was carried out for genetic polymorphisms that failed genotyping. After excluding two genetic polymorphisms with a minor allele frequency (MAF) <0.01, call rate <90%, or Hardy–Weinberg equilibrium P value <0.001, 101 polymorphisms from 23 genes were finally selected for analysis (Supplementary Table S1 , available at https://doi.org/10.1016/j.esmoop.2021.100236 ).
5
Genotyping of PE/PPE and non-PE/PPE SNPs
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PCR and extension primers were designed for 60 PE/PPE and 60 randomly selected non-PE/PPE SNPs using MassArray Assay Design 3.1 software (Sequenom, San Diego, CA). Five were excluded due to difficult sequences. PCRs contained, in a volume of 5 µl, 1 pmol of the corresponding primers, 10 ng genomic DNA, and HotStar reaction Mix (Qiagen) in 384-well plates. PCR conditions were as follows: 94 °C for 15 min, followed by 40 cycles of 94 °C (20 s), 56 °C (30 s), 72 °C (60 s), and a final extension of 72 °C for 3 min. In the primer extension procedure, each sample was denatured at 94 °C, followed by 40 cycles of 94 °C (5 s), 52 °C (5 s), 72 °C (5 s). The mass spectrum from time-resolved spectra was retrieved by using a MassARRAY mass spectrometer (Sequenom), and each spectrum was then analyzed using SpectroTYPER software (Sequenom) to perform the genotype calling. After analyzing the genotype profiles, the clustering patterns of five SNPs could not be used to correctly perform genotype calling, and the data of 110 SNPs (57 PE/PPE and 53 non-PE/PPE) were finally used in the following analyses.
6
Genotyping of Peripheral Blood DNA
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Genomic DNA was extracted from peripheral blood leucocytes by proteinase K digestion followed by phenol-chloroform method. Genotyping was done using MassARRAY system (Sequenom, San Diego, CA, USA) following the manufacturer's instructions as published elsewhere [38 ]. SpectroTYPER software (Sequenom) automatically called the genotypes and only conservative and moderate calls were accepted for the study. Ten percent of the samples genotyped were replicated and discordance rate observed was less than 0.4% for the replicated samples. All the variants genotyped had call rate ranging between 90%–99%.
7
Genotyping ADARB1 and ADARB2 Genes using Sequenom
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SNPs in the ADARB1 and ADARB2 genes were genotyped using the Sequenom MassARRAY system 4 platform, and Typer 4.0 software was used to carry out all genotyping work (MALDI-TOF; MassARRAY system, Sequenom Inc., San Diego, CA, USA). The primers for PCR and iPLEX reactions were designed using the online Assay design suite 1.0 Sequenom software (available at: www.mysequenom.com/Home ) and obtained from IDT (see Table 4 ) (Integrated DNA Technologies, Carolville, Iowa, USA). PCR and extension reactions were performed in a 96-well plate according to the manufacturer’s instructions, using Sequenom reagents. Completed genotyping reactions were spotted in nanoliter volumes onto a matrix arrayed silicon chip with 96 elements (Sequenom SpectroCHIP) using the MassARRAY Nanodispenser. Spectro CHIPs were analyzed using the Bruker Autoflex MALDI-TOF mass spectrometer and the spectra were processed using the SpectroTYPER software (Sequenom) to yield genotypes.
8
Genotyping Protocol for SNP Analysis
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Peripheral blood (3-5 mL) was collected using tubes coated with ethylenediaminetetraacetic acid (EDTA) and stored at -80°C. Genome DNA was extracted using an EZNA TM Blood DNA Midi Kit (Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer instructions. DNA was stored at -80°C for SNP analysis. Genotyping was carried out on a MassARRAY platform (Sequenom, San Diego, CA, USA). The target fragments were augmented by polymerase chain reaction (PCR). All the products were treated with shrimp alkaline phosphatase. Single nucleotide extension was then carried out using iPLEX enzyme (Sequenom). The samples were spotted onto a 384-well spectroCHIP nanodispenser (Sequenom) and analyzed using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MassARRAY system in the fully automated mode. Genotypes were automatically identified using SpectroTYPER software (Sequenom).
9
Genotyping of MMP Gene Polymorphisms
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Human genomic DNA was extracted from peripheral blood samples using an automated nucleic acid extraction system (Lab-Aid 820, Xiamen Zeesan Biotech, Xiamen, China) and all samples were stored in Tris-ethylenediaminetetraacetic acid buffer. Polymerase chain reaction (PCR) amplification was performed on an ABI GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). Reactions included an initial denaturation stage at 95°C for 2 min, followed by 45 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s, before a final extension step at 72°C for 5 min. Shrimp alkaline phosphatase was then used to inactivate unincorporated nucleotides. Genotyping was conducted with an iPLEX Gold Assay using the MassARRAY platform (Sequenom, San Diego, CA, USA). We carried out a primer extension reaction using Mass-EXTEND primers, the products of which were purified on resin and then spotted using a Sequenom Nanodispenser onto a SpectroCHIP to crystalize. The polymorphisms tested were MMP-1 rs2075847, MMP-2 rs2285053, MMP-9 rs3918250, MMP-12 rs652438, and MMP-13 rs640198. Primer sequences used to genotype the five MMP polymorphisms are shown in Table 1. Data were analyzed with SpectroTYPER software (version 4.0; Sequenom).
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