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Massarray compact analyzer

Manufactured by Agena
Sourced in Germany, United States

The MassARRAY Compact Analyzer is a laboratory instrument designed for high-throughput genetic analysis. It utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry technology to perform accurate and efficient genotyping and mutation detection.

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4 protocols using massarray compact analyzer

1

Comparative Genomic Typing of Bacterial Isolates

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Raw sequence chromatograms were examined using Chromas version 2.6.2 (Technelysium Pty Ltd, Brisbane, Australia) before consensus sequences were created using BioEdit Sequence Alignment Editor version 7.2.5 [15 ]. spa types were assigned using the online spa type finder/identifier software, http://spatyper.fortinbras.us/ (Fortinbras Research). spa types were further confirmed using the Ridom spa Server, https://www.spaserver.ridom.de/ (Ridom GmbH, Würzburg, Germany) [16 (link)]. For MLST loci, consensus sequences were aligned using the online alignment tool, MAFFT version 7 https://mafft.cbrc.jp/alignment/server/. Sequence types were assigned on the MLST database, www.mlst.net [14 (link)], using the ‘Exact or Nearest Match’ option. The SpectroAcquire program was used for data acquisition on the MassARRAY Compact Analyzer (Agena Bioscience, Hamburg, Germany) and detection parameters were set at 10 laser shots per raster position with a threshold of 5 good spectra per sample pad. In estimating the accuracy of iPex MassARRAY, all isolates were listed with their SNP genotypes and corresponding spa and MLST sequence types (STs). Spa and MLST classifications were used as the reference methods. The accuracy of iPlex MassARRAY was determined as the proportion of isolates whose SNP genotypes corresponded to particular spa/MLST types without ambiguity.
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2

Genetic Variations in CYP2C8 and CYP2C9

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For the selection of tag single nucleotide polymorphisms (SNPs), Haploview 4.2 software was used. The SNPs were obtained from the HapMap phase III JPT+CHB database using a threshold of r 2 ≥0.5. In addition, the SNPs were located in CYP2C8 and CYP2C9 gene regions including 30 kb upstream and 30 kb downstream. We selected 9 tag SNPs for CYP2C8 and 10 tag SNPs for CYP2C9. The tag SNPs have a minor allele frequency (MAF) 0.05. In order to cover the most part of the physical location of the genes, the SNP were located in intron or non-coding region part, so the classic variation in exome region like CYP2C8*2 or CYP2C8*3 were not analysis in our study. All SNPs were genotyped by MassArray Compact Analyzer (Agena Bioscience, San Diego, CA, USA) with matrix-assisted laser desorption ionisation-time of ight mass spectrometry. Then, we tested the data quality in our sample; all SNPs should have had a MAF more than 0.01. All of the SNPs passed quality control with call rates 90% and concordance rates 99%.
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3

Mosaic Mutation Detection using iPLEX and MALDI-TOF MS

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The mosaic mutation was confirmed by using iPLEX and MALDI-TOF MS (Agena Bioscience, Hamburg, Germany). The iPLEX reaction was performed according to the standard protocol recommended by the system supplier (21 (link)). The homogeneous MassEXTEND (hME) and iPLEX process reley on a small volume PCR amplifying the target regions including the SNP position in a multiPLEX fashion. The basic principle of hME and iPLEX reaction is identical. Both methods use a third, so-called MassEXTEND primer, which anneals directly adjacent to the SNP position. In an enzymatic primer extension reaction, this primer will be elongated. During that process the allele-specific analytical products are generated. The products differ by mass according to the incorporated bases. Primers were designed: ACGTTGGATGCTGTGAGCACCAATTTGGAC (PHEX-ex21_PCR1) and ACGTTGGATGTTCTCTTCTAGGTGAGGTGC (PHEX-ex21_PCR2), with the tag-sequences in italics. For the iPLEX-reaction the primer sequences were: ACAGACCAGAAGCTGCC (left) and CCAATTTGGACTTGTTCTC (right). The sample carrier was introduced into the mass spectrometer (MassARRAY Analyzer Compact, Agena Bioscience) and data are fully automatically acquired and analyzed in a real-time setting and revised using Typer software (Agena Bioscience).
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4

MALDI-TOF Genotyping Protocol

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The samples on the SpectroChip were analyzed on the MSArray workstation (Agena Bioscience) (29 (link)) with a MALDI-TOF mass spectrometer (MassArray analyzer compact, Agena Bioscience) containing a Class I nitrogen laser emitting 40–90 microjoules at 337 nm. A calibration spectrum was acquired for each chip with the 3-point calibrators provided with the system. Spectral measurements were acquired for the center and 4 other positions around each matrix pad. We used MassArray Typer4.0 (Agena Bioscience) software to analyze the data from the spectral peaks to determine the genotyping call for each sample.
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