Ion pgm hi q sequencing kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The Ion PGM Hi-Q Sequencing Kit is a laboratory equipment product designed for DNA sequencing. It provides the necessary reagents and consumables for performing DNA sequencing using the Ion PGM system. The kit enables the sequencing of genetic samples and the analysis of their DNA sequences.

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Ion PGM (71 citations)

69 protocols using ion pgm hi q sequencing kit

Ion Torrent PGM Sequencing of Barcoded Samples

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All barcoded samples were sequenced using the Ion PGM™ Hi-Q™ Sequencing Kit (Life Technologies) in an Ion Torrent PGM instrument (Life Technologies) with Ion 318™ v2 chips (Life Technologies).
Chip loading procedure was performed according to the user guide for the Ion PGM™ Hi-Q™ Sequencing Kit (Life Technologies). A maximum of 16 samples were loaded on a single chip per sequencing run.

Brandariz L., Arriba M., García J.L., Cano J.M., Rueda D., Rubio E., Rodríguez Y., Pérez J., Vivas A., Sánchez C., Tapial S., Pena L., García-Arranz M., García-Olmo D., Urioste M., González-Sarmiento R, & Perea J. (2018). Differential clinicopathological and molecular features within late-onset colorectal cancer according to tumor location. Oncotarget, 9(20), 15302-15311.

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Mitochondrial DNA Sequencing Protocol

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PCR products were purified using QIAquick PCR purification kit (QIAGEN). Ion Torrent deep sequencing libraries were constructed from bisulfite-converted DNA using the KAPA Library Preparation Kit (Kapa Biosystems), quantified using the QIAxcel Advanced System (QIAGEN), and templated using the Ion PGM Template OT2 200 kit (Thermo Fisher). The libraries were sequenced using an Ion PGM Sequencing HiQ Kit with Ion 316 v2 Chips on the Ion Torrent PGM (Thermo Fisher), which generated non-directional ~200 nt length reads, 1500–7500 reads per library in the fastq format. Reads were aligned to human or mouse mtDNA reference sequences (hg38 and mm10, respectively) using the Bismark aligner [25 (link)] with bowtie2 option. Bisulfite conversion efficiency was evaluated based on cytosine conversion rate (C versus T) at non-CpG sites determined after visualization of the aligned reads (bam format) using the Integrative Genomics Viewer (IGV) [18 (link)].

Owa C., Poulin M., Yan L, & Shioda T. (2018). Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection. PLoS ONE, 13(2), e0192722.

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Ion Torrent PGM Library Sequencing

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The library pool was clonally amplified in an emulsion PCR reaction using Ion Sphere Particles (ISPs) in the One Touch 2 Instrument. Subsequently, template-positive ISPs were enriched using the Ion One Touch ES with the Ion PGM Hi-Q OT2 kit following manufacturer´s protocol. Enriched template-positive ISPs were subjected to sequencing on the Ion Torrent Personal Genome Machine (PGM) on a 318v2 Ion Chip using Ion PGM Sequencing Hi-Q kit (all kits from ThermoFisher Scientific).

Simarro J., Murria R., Pérez-Simó G., Llop M., Mancheño N., Ramos D., de Juan I., Barragán E., Laiz B., Cases E., Ansótegui E., Gómez-Codina J., Aparicio J., Salvador C., Juan Ó, & Palanca S. (2019). Development, Implementation and Assessment of Molecular Diagnostics by Next Generation Sequencing in Personalized Treatment of Cancer: Experience of a Public Reference Healthcare Hospital. Cancers, 11(8), 1196.

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Ion PGM-based Sequencing of Circularized DNA

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PCR was performed with the following reaction setup: 1 μl of purified and circularized DNA solution, 16.99 μl of Nuclease-Free water, 2.5 μl of 10× High Fidelity PCR Buffer, 2.2 μl of 2.5 mM dNTP, 1.2 μl of 50 mM MgSO4, 0.11 μl of 5 unit/μl Platinum Taq, 0.5 μl of 10 μM A primer, 0.5 μl of 10 μM P1-DPB1-No1~No12 in shown in Fig. 5c and Additional file 6: Table S3. The following cycling conditions were used: 1 cycle of 94 °C for 30 s, 40 cycles of 94 °C for 30 s, 65 °C for 30 s, and 68 °C for 1 min. After mixing each PCR product, gel extraction (range from 200 bp to 500 bp) was performed using the MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). The concentration of the purified products was measured using the Quanti-iT™ dsDNA Assay Kit, Broad range. Using 2.5 μl of 100 pM purified products, we prepared sequencing templates with Ion One Touch™ 2 (Thermo Fisher Scientific) and Ion One Touch™ ES (Thermo Fisher Scientific). Sequencing was performed using the Ion PGM™ Sequencing Hi-Q Kit (Thermo Fisher Scientific).

Segawa H., Kukita Y, & Kato K. (2017). HLA genotyping by next-generation sequencing of complementary DNA. BMC Genomics, 18, 914.

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Somatic Mutation Detection via NGS

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Several methods for detecting somatic mutations are currently available. NGS, considered to be an optimum method for mutation detection, was conducted employing the Ion Torrent PGM technology (ThermoFisher Scientific). The Oncomine Solid Tumor DNA kit was used for DNA analysis (ThermoFisher Scientific). This assay simultaneously screens hotspots mutations in 22 genes (included PIK3CA). For generating the DNA barcoded libraries, a multiplex PCR amplification of 10 ng of genomic DNA was performed. Sequencing was performed on the Ion Torrent PGM system on a 318v2 Ion Chip using Ion PGM Sequencing Hi-Q kit (Thermo Fisher Scientific). Data from sequencing runs were transferred to the Torrent Server, alignment to the hg19 human reference genome and variant calling was performed by the Ion Torrent Suite Software 5.4 (Thermo Fisher Scientific). In addition, all identified variants, such as PIK3CA mutations in exon 9 and 20 (p.E545K and p.H1047L), were visually checked using the Integrative Genomics Viewer (IGV) software.

Martorell S., Palanca S., Maquieira Á, & Tortajada-Genaro L.A. (2018). Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene. Analytical biochemistry, 544.

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Gut Microbiome Analysis via 16S rRNA Sequencing

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DNA was extracted from fecal samples using a PowerSoil DNA Extraction Kit (MO BIO Laboratories, Carlsbad, CA). Polymerase chain reaction (PCR) was performed using a primer set (784F: 5′‐AGGATTAGATACCCTGGTA‐3′ and 1061R: 5′‐CRRCACGAGCT‐GACGAC‐3′) targeting the V5‐V6 region of the 16S rRNA genes with KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Woburn, MA). DNA libraries were prepared using an Ion Fragment Library Kit (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Sequencing was performed using two 318 chips and Ion PGM Sequencing Hi‐Q Kits on an Ion PGM sequencer (both, Life Technologies). The resulting sequences were analyzed with Quantitative Insights into the Microbial Ecology pipeline.

Muratsu A., Ikeda M., Shimizu K., Kameoka S., Motooka D., Nakamura S., Matsumoto H., Ogura H, & Shimazu T. (2022). Dynamic change of fecal microbiota and metabolomics in a polymicrobial murine sepsis model. Acute Medicine & Surgery, 9(1), e770.

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Fecal Microbiome Changes in Sepsis

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Fecal samples were collected on day 0 (before CLP) and days 1, 3, and 7 after CLP to determine the microbiome. DNA was extracted from fecal samples using a PowerSoil DNA Extraction Kit (MO BIO Laboratories, Carlsbad, Calif). Polymerase chain reaction (PCR) was performed using a primer set (784F: 5′-AGGATTAGATACCCTGGTA-3′ and 1061R: 5′-CRRCACGAGCTGACGAC-3′) targeting the V5-V6 region of the 16S rRNA genes with KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Woburn, Mass) (12) . DNA libraries were prepared using an Ion Fragment Library Kit (Life Technologies, Gaithersburg, Md) according to the manufacturer's instructions. Sequencing was performed using two 318 chips and Ion PGM Sequencing Hi-Q Kits (Life Technologies) on an Ion PGM sequencer (Life Technologies). The resulting sequences were analyzed with the quantitative insights into the microbial ecology pipeline (13) .

Ikeda M., Shimizu K., Ogura H., Kurakawa T., Umemoto E., Motooka D., Nakamura S., Ichimaru N., Takeda K., Takahara S., Hirano S.I, & Shimazu T. (2018). Hydrogen-Rich Saline Regulates Intestinal Barrier Dysfunction, Dysbiosis, and Bacterial Translocation in a Murine Model of Sepsis. Shock (Augusta, Ga.), 50(6).

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Ion Torrent PGM DNA Sequencing

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Approximately 1 μg of purified WGA DNA was used for library construction by the Ion Xpress Plus gDNA Fragment Library Preparation Kit Set (Life technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. The quantity of library was determined using Qubit dsDNA HS assay kits (Life technologies) with Qubit fluorometers (Life technologies). The template-positive Ion Sphere Particles were generated using Ion PGM Hi-Q Template Kits (Life technologies) with the Ion OneTouch 2 Instrument (Life technologies) and then enriched with the Ion OneTouch ES Instrument (Life technologies). Sequencing was performed on the Ion Torrent PGM Instrument (Life technologies) platform with the Ion PGM Hi-Q Sequencing Kit and Ion 316 chip (Life technologies). The sequencing data analysis was performed by using the cloud-based the Ion Reporter^TM Server System (https://ionreporter.thermofisher.com/ir/).

Ma G.C., Lin W.H., Huang C.E., Chang T.Y., Liu J.Y., Yang Y.J., Lee M.H., Wu W.J., Chang Y.S, & Chen M. (2019). A Silicon-based Coral-like Nanostructured Microfluidics to Isolate Rare Cells in Human Circulation: Validation by SK-BR-3 Cancer Cell Line and Its Utility in Circulating Fetal Nucleated Red Blood Cells. Micromachines, 10(2), 132.

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Ion Torrent Sequencing Library Preparation

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Sequencing libraries were quantified with quantitative polymerase chain reaction (qPCR) with the Ion Library Quantitation Kit (Cat # 4,468,802, Life Technologies) and diluted to 100 pM. The diluted sequencing libraries (10 pM) were then amplified on Ion Sphere^TM Particles (ISPs) and enriched for template-positive ISPs using the Ion PGM Template OT2 200 Kit (Cat # 4,480,974), Ion One Touch 2 instrument, and Ion One Touch ES. The Ion Sphere Quality Control Kit (Cat # 4,468,656, Life Technologies) was used to determine the fraction of template-positive ISPs. Enriched ISPs were then sequenced using the Ion PGM Hi-Q sequencing Kit (Cat # A25592, Life Technologies) and the 318 Chip Kit v2 (Cat # 4,484,354, Life Technologies). Ion Torrent sequencing results were deposited in the NCBI Sequence Read Archive with study accession number SRP067259.

Collins D.W., Gudiseva H.V., Trachtman B., Bowman A.S., Sagaser A., Sankar P., Miller-Ellis E., Lehman A., Addis V, & O'Brien J.M. (2016). Association of primary open-angle glaucoma with mitochondrial variants and haplogroups common in African Americans. Molecular Vision, 22, 454-471.

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Ion PGM Semiconductor Sequencing Protocol

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Semiconductor sequencing on the Ion PGM^TM detects the change in pH when the proton (H+) is released during nucleotide incorporation. The sequencing was conducted on the Ion 318™ Chip v2 using the Ion PGM™ Hi‐Q™ Sequencing Kit (six barcoded samples per chip) coupled with the PGM^TM sequencer (Life Technologies, a part of Thermo Fisher Scientific Inc.). A chlorite cleaning, followed by 18 MΩ water wash (Thermo Fisher Scientific, Millipore, MA) was performed prior to the initialization of the Ion PGM™ System.

Chaitanya L., Ralf A., van Oven M., Kupiec T., Chang J., Lagacé R, & Kayser M. (2015). Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine. Human Mutation, 36(12), 1236-1247.

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