Ion pgm hi q chef kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion PGM™ Hi-Q™ Chef Kit is a laboratory equipment product designed for DNA sequencing. It provides the necessary reagents and consumables to automate the sample preparation process for Ion Torrent sequencing platforms.

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Ion Chef (118 citations) Ion PGM (71 citations) Ion PGM Hi-Q View Chef Kit (7 citations)

12 protocols using ion pgm hi q chef kit

Amplification and Quantification of Barcoded Libraries

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Libraries were amplified on an Applied Biosystems Veriti^™ 96 well Thermal Cycler (Thermo Fisher Scientific) in a total reaction volume of 130 μl, containing 100 μl of Platinum^®PCR SuperMix High Fidelity, 5 μl of Library Amplification Primer Mix (both provided by the IPFL kit) and 25 μl of unamplified library. The cycling program suggested on the IPFL kit protocol was applied. Amplified libraries were purified with Agencourt^® AMPure^® XP Kit for DNA purification (Beckman Coulter, Beverly, Massachusetts, USA) on a DynaMag^™-2 magnet magnetic rack (Thermo Fisher Scientific) following the procedure proposed by the manufacturer. Agilent 2100 Bioanalyzer (Agilent Genomics) was used to determine the molar concentration of each barcoded library. Three equimolar pools of barcoded libraries were prepared: barcoded libraries from SUR-1 to SUR-6 (Pool 1), from SUR-7 to SUR-12 (Pool 2) and from SUR-13 to SUR-16 (Pool 3) were pooled together. The three pools were quantified on Agilent 2100 Bioanalyzer (Agilent Genomics) or Library TaqManTM Quantitation Kit (Thermo Fisher Scientific) following the procedure proposed by the manufacturer, and then diluted as proposed by the Ion PGM^™ Hi-Q^™ Chef Kit (Thermo Fisher Scientific).

Giusti A., Armani A, & Sotelo C.G. (2017). Advances in the analysis of complex food matrices: Species identification in surimi-based products using Next Generation Sequencing technologies. PLoS ONE, 12(10), e0185586.

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Ion Torrent Sequencing Workflow

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Six uniquely barcoded library samples were pooled per run for sequencing on an Ion 318 v2 chip (ThermoFisher Scientific). The Ion Chef System (ThermoFisher Scientific) was used with the Ion PGM Hi-Q Chef Kit (ThermoFisher Scientific) for fully automated template preparation and Ion 318 v2 chip loading. Single-end sequence analysis was performed using the Ion PGM Hi-Q Sequencing Kit on the Ion Torrent Personal Genome Machine (Ion PGM) (ThermoFisher Scientific) for 200 base-read sequencing.

Park J., Yoo H.M., Sul H.J., Shin S., Lee S.W, & Kim J.G. (2019). Genetic Characterization of Molecular Targets in Korean Patients with Gastrointestinal Stromal Tumors. Journal of Gastric Cancer, 20(1), 29-40.

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Ion PGM Hi-Q Chef Kit for NGS

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The Ion PGM^™ Hi-Q^™ Chef Kit (Thermo Fisher Scientific) was utilized to prepare template-positive Ion Sphere^™ Particles (ISP) and to load three Ion 314^™ v2 BC sequencing chips (Chip 1 for Pool 1, Chip 2 for Pool 2 and Chip 3 for Pool 3) on Ion Chef^™ System (Thermo Fisher Scientific) following the manufacturer protocol.

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Amplicon Sequencing for Inherited Disease

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For the amplicon sequencing, the Ion AmpliSeq Library Kit 2.0 (catalog # 4475345, ThermoFisher, Waltham, MA) and AmpliSeq Inherited Disease Panel (catalog# 4477686, ThermoFisher, Waltham, MA) were used in accordance with the manufacturer’s protocol. DNA from each genome was amplified in three separate primer pools, then these PCR products were combined for barcoding and library preparation. The concentration of the final library was measured with the Ion Library TaqMan Quantification Kit (catalog #4468802, ThermoFisher, Waltham, MA), then two libraries were adjusted to a concentration of 40 picomol/L and combined before chip loading. 318v2 BC chips (catalog #4488146, ThermoFisher, Waltham, MA) were loaded using the Ion Chef and then sequenced on the Personal Genome Machine using the Ion PGM Hi-Q Chef kit (catalog # A25948, ThermoFisher, Waltham, MA).

Cleveland M.H., Zook J.M., Salit M, & Vallone P.M. (2018). Determining Performance Metrics for Targeted Next Generation Sequencing Panels Using Reference Materials. The Journal of molecular diagnostics : JMD, 20(5), 583-590.

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Targeted Sequencing of Hotspot Mutations

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Library preparation was carried out using the Oncomine Assay™ (comprising the DNA Oncomine™ Focus Assay) (Thermo Fisher Scientific) following the manufacturer’s instructions, using a total of 10 ng input DNA per sample (minimum 0.83 ng/μL sample DNA concentration). A maximum of seven DNA samples were prepared per run on an Ion 318™ v2 chip (Thermo Fisher Scientific, catalog no. 4488150). The DNA panel can identify hotspot mutations in the following genes: AKT1, ALK, AR, BRAF, CDK4, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FGFR2, FGFR3, GNA11, GNAQ, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, PIK3CA, RAF1, RET, ROS1, and SMO; and K-RAS was the focus of this validation. Template preparation was performed on the Ion Chef System (Thermo Fisher Scientific, Waltham, MA, USA) using the Ion PGM Hi-Q Chef Kit and/or the Ion One Touch™ 2 System using the Ion PGM Template OT2 200 Kit. Sequencing was performed using the Ion PGM Hi-Q Sequencing Kit on the Ion Torrent Personal Genome Machine (Ion PGM).

Aran V., Zalis M., Montella T., de Sousa C.A., Ferrari B.L, & Gil Ferreira C. (2021). Evaluation of KRAS Concomitant Mutations in Advanced Lung Adenocarcinoma Patients. Medicina, 57(10), 1039.

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Sequencing and Analysis of Ancestry Panels

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Thirty-five Precision ID Ancestry Panel libraries were sequenced in pools containing 18 libraries (one library was not used in this study), which had been pooled in equimolar concentrations according to Qubit, TapeStation, or ABI7500 (IonLibQuant assay) quantifications. Each pool had a total library concentration of 50 pM. Template preparation consisting of emulsion PCR, enrichment of beads containing template, and chip loading was performed with the Ion Chef instrument and the Ion PGM Hi-Q Chef Kit according to the manual (Thermo Fisher Scientific). The loaded sequencing chips were placed onto the Ion PGM™ instrument (Thermo Fisher Scientific) together with Ion PGM Hi-Q Chef 400 Supplies Kit (Thermo Fisher Scientific) and sequenced for 500 cycles according to the manual. Sequence analysis was done using the Torrent Suite Software v.4.4.2 with the HID_SNP_GenoTyper v. 4.2 plugin. Linear regression and two-sided F test were performed using the “lm” application in R 3.3.0 to test for correlations between concentration estimates and library coverage using data from all 35 libraries and α = 0.05.

Hussing C., Kampmann M.L., Mogensen H.S., Børsting C, & Morling N. (2018). Quantification of massively parallel sequencing libraries – a comparative study of eight methods. Scientific Reports, 8, 1110.

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Ion PGM Library Preparation and Sequencing

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Before sequencing, the template preparation for library pools was performed either with Ion OneTouch 2 system (Thermo Fisher Scientific) using the Ion PGM™ Hi-Q™ OT2 Kit (Thermo Fisher Scientific) or Ion Chef system (Thermo Fisher Scientific) using the Ion PGM™ Hi-Q™ Chef Kit following the kit protocols. 2 µl of each 10 pM library was used for library pool, and 15–20 libraries were pooled together. 20 µl of a library pool was mixed with 5 µl of nuclease-free water and added to the amplification solution for template preparation. After the template preparation, the quality of resulting ion spheres was checked with Qubit 3.0 fluorometer (Thermo Fisher Scientific). Following quality check, the ion spheres were loaded on an Ion 318™ Chip (Thermo Fisher Scientific) and sequenced with Ion PGM system using Ion PGM Hi-Q Sequencing kit (Thermo Fisher Scientific) according to the protocol provided with the kit.

Youssef O., Lahti L., Kokkola A., Karla T., Tikkanen M., Ehsan H., Carpelan-Holmström M., Koskensalo S., Böhling T., Rautelin H., Puolakkainen P., Knuutila S, & Sarhadi V. (2018). Stool Microbiota Composition Differs in Patients with Stomach, Colon, and Rectal Neoplasms. Digestive Diseases and Sciences, 63(11), 2950-2958.

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Ion Torrent PGM Sequencing Protocol

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A total of six uniquely barcoded library samples were pooled for sequencing per run on an Ion 318™ v2 chip (Thermo Fisher Scientific, Inc.). The Ion Chef™ System (Thermo Fisher Scientific, Inc.) was applied using the Ion PGM™ Hi-Q™ Chef Kit for fully automated template preparation and Ion 318™ v2 chip loading. Single-end sequence analysis was performed using the Ion PGM™ Hi-Q™ Sequencing Kit on the Ion Torrent PGM™ (Thermo Fisher Scientific, Inc.) for 200 base-read sequencing.

Park J., Lee S.I., Shin S., Hong J.H., Yoo H.M, & Kim J.G. (2020). Genetic profiling of somatic alterations by Oncomine Focus Assay in Korean patients with advanced gastric cancer. Oncology Letters, 20(5), 129.

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Ion AmpliSeq Cancer Hotspot Sequencing

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Target regions were amplified using the Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies). Template DNA was prepared using the Ion PGM Hi-Q Chef Kit (Life Technologies) and sequencing was run on the Ion PGM using the Ion 314 chip. Reads were aligned to the hg19 reference and the analysis was carried out using the Ion Torrent Variant Caller Plugin and the Ion Reporter (Life Technologies).

Kasai F., Hirayama N., Ozawa M., Satoh M, & Kohara A. (2018). HuH-7 reference genome profile: complex karyotype composed of massive loss of heterozygosity. Human Cell, 31(3), 261-267.

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Ion Torrent Amplicon Sequencing Protocol

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For each single genome amplicon, 100 ng of second round PCR product was enzymatically sheared to 400 bp followed by barcoding using the Ion Xpress Plus Fragment Library & Ion Xpress Barcode Adapters kits (LifeTechnologies, ThermoFisher Scientific). Quantification was performed using a 2100 Bioanalyzer (DNA 1000 kit, Agilent Technologies, Sunnyvale, CA). DNA size-selection was performed using Blue Pippin 2% dye free cassette with internal standard marker V1 (Sage Science). The size-selected product was equalized using the Ion Equalizer kit (LifeTechnologies, ThermoFisher Scientific) following manufacturer’s instructions. All purifications used Agencourt AMPure XP Reagent (Beckman Coulter).
Emulsion PCR (ePCR) and enrichment for 400 bp sheared product used the Ion OneTouch 400 bp Template kit (LifeTechnologies, Carlsbad, CA) on the OneTouch and ES instruments. Sequencing was carried out using the Ion PGM 400 kit and Ion 316 chip v2 on the IonTorrent PGM platform (LifeTechnologies, Carlsbad, CA), following manufacturer’s instructions. For scalability experiments, the ePCR/enrichment of libraries derived from 43 different amplicons were carried out on the Ion Chef instrument with Ion PGM Hi-Q Chef kit (LifeTechnologies, Carlsbad, CA) and were sequenced on Ion 318 chips v2 BC.

Kijak G.H., Sanders-Buell E., Pham P., Harbolick E.A., Oropeza C., O’Sullivan A.M., Bose M., Beckett C.G., Milazzo M., Robb M.L., Peel S.A., Scott P.T., Michael N.L., Armstrong A.W., Kim J.H., Brett-Major D.M, & Tovanabutra S. (2019). Next-generation sequencing of HIV-1 single genome amplicons. Biomolecular Detection and Quantification, 17, 100080.

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