Pyro mark id instrument

Manufactured by Qiagen

Sourced in Germany

The PyroMark ID is a laboratory instrument designed for DNA pyrosequencing analysis. It performs automated, high-throughput analysis of short DNA sequences. The instrument utilizes a sequencing-by-synthesis approach to determine the nucleotide sequence of DNA samples.

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Lab products found in correlation

Q cpg software v1, by Biotage (2 mentions) Pyromark q96, by Qiagen (2 mentions) Pyromark gold enzyme, by Qiagen (2 mentions) Pyromark q96 id, by Qiagen (1 mentions) Pyrogold sqa reagent kit, by Qiagen (1 mentions) Psq hs 96 system, by Qiagen (1 mentions) Pyromark q96 cpg line 1 kit, by Qiagen (1 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions)

5 protocols using pyro mark id instrument

Rapid Genotyping of I38X Influenza Variant

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In order to perform rapid genotyping for the substitution I38X of the influenza virus cap dependent endonuclease (PA), which is associated with reduced susceptibility to baloxavir marboxil, a pyrosequencing method was designed and established, which is described in detail in the online protocol Pyrosequencing analysis for molecular marker I38X for reduced susceptibility of influenza A viruses to baloxavir marboxil [DOI: dx.doi.org/10.17504/protocols.io.n92ldzkpov5b/v1].
Primers were designed using PyroMark Assay Design version 2.0.2.5 (Qiagen, Germany) with A/Kansas/14/2017 (GISAID Acc.No EPI_ISL_292575) as reference; the reverse primer was biotinylated. PCR was validated using A(H1N1)pdm09 and A(H3N2) influenza A virus cell-cultured isolates. The 100 bp amplicons were analyzed on 1.5% agarose gels. Pyrosequencing was performed with up to 96 samples in parallel at 28°C with the PyroMark ID Instrument using PyroMark Q96 ID (Qiagen, Germany) enzyme and substrate mixture using SQA mode with 10fold CTGA and SNP analysis as described elsewhere.19 (link)

Oh D.Y., Milde J., Ham Y., Ramos Calderón J.P., Wedde M., Dürrwald R, & Duwe S.C. (2023). Preparing for the Next Influenza Season: Monitoring the Emergence and Spread of Antiviral Resistance. Infection and Drug Resistance, 16, 949-959.

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Quantifying Global LINE-1 Methylation

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LINE-1 methylation was carried out on the placental, maternal and cord blood DNA of all cases (Figure 1) in order to measure the global LINE-1 methylation pattern in the placenta to compare it with adult and cord blood, and to assess possible relationships with the birth-weight percentile.
LINE-1 methylation analyses were performed as previously described [41 (link)]. In brief, the PyroMark Q96 CpG LINE-1 kit (QIAGEN, Hilden, Germany) was used to amplify LINE-1 sequences from 20 ng of bisulfite-converted DNA and to quantify methylation levels at four CpG sites. Quantitative DNA methylation analyses were performed using pyrosequencing, using the Pyro Mark ID instrument (QIAGEN, Hilden, Germany), equipped with PSQ HS 96 System, and PyroGold SQA reagent kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Raw data were analyzed using the Q-CpG software v1.0.9 (Biotage AB, Sweden), which calculates the ratio of converted C’s (T’s) to unconverted C’s at each CpG, giving the percentage of methylation [50 (link)]. Reported methylation values are the mean between at least two independent PCR and pyrosequencing experiments.

Rondinone O., Murgia A., Costanza J., Tabano S., Camanni M., Corsaro L., Fontana L., Colapietro P., Calzari L., Motta S., Santaniello C., Radaelli T., Ferrazzi E., Bosari S., Gentilini D., Sirchia S.M, & Miozzo M. (2021). Extensive Placental Methylation Profiling in Normal Pregnancies. International Journal of Molecular Sciences, 22(4), 2136.

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Influenza Virus Genomic Composition Analysis

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Genomic composition of the reassortant influenza viruses was assessed by pyrosequencing as described previously (Shcherbik et al., 2014b (link)). Briefly, cDNA was produced from viral RNA with gene specific sets of primers in which either the forward or reverse primer was biotinylated. The biotinylated amplicons were bound to streptavidin-coated beads and subjected to denaturation and washing to generate single stranded DNAs, which were then annealed with pyrosequencing primer, designed to detect the strain-specific signature nucleotides. Pyrosequencing reactions were performed with the PyroMark Gold enzyme, substrate and nucleotides using the PyroMark ID instrument (Qiagen). Sequence results were obtained in the form of pyrograms and analyzed using visual interpretation and the PyroMark Q96 software (Qiagen).

Shcherbik S., Pearce N., Kiseleva I., Larionova N., Rudenko L., Xu X., Wentworth D.E, & Bousse T. (2015). Implementation of new approaches for generating conventional reassortants for live attenuated influenza vaccine based on Russian master donor viruses. Journal of virological methods, 227, 33-39.

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Quantitative DNA Methylation Analysis

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Total DNA was extracted from LCLs using the QIAamp^® DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Two independent quantitative methylation experiments of IGF2, H19, GNAS, GNAS-AS1, MEST and PEG10 DMRs were performed by pyrosequencing using the Pyro Mark ID instrument (Qiagen, Hilden, Germany). Raw data were analyzed using the Q-CpG software v1.09 (Biotage Sweden AB). Details on the genomic positions, set-up and protocol were previously described [45 (link),46 (link)].

Pileggi S., La Vecchia M., Colombo E.A., Fontana L., Colapietro P., Rovina D., Morotti A., Tabano S., Porta G., Alcalay M., Gervasini C., Miozzo M, & Sirchia S.M. (2021). Cohesin Mutations Induce Chromatin Conformation Perturbation of the H19/IGF2 Imprinted Region and Gene Expression Dysregulation in Cornelia de Lange Syndrome Cell Lines. Biomolecules, 11(11), 1622.

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Influenza Virus Genomic Composition Analysis

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