Ion 510 ion 520 ion 530 kit chef

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion 510 & Ion 520 & Ion 530 Kit—Chef is a set of laboratory equipment designed for DNA and RNA sequencing. It provides the necessary components for sample preparation, template generation, and sequencing on the Ion Torrent semiconductor sequencing platform.

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5 protocols using ion 510 ion 520 ion 530 kit chef

Targeted Resequencing of Cancer Genes

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Targeted resequencing was performed by Next Generation Sequencing (Ion GeneStudio S5 prime, Thermo Fisher Scientific) using an AmpliSeq Custom Panel comprising hotspot regions in STAT3, KRAS, NRAS, BRAF, and the complete coding sequence of TP53 and TET2 (Supplementary Data 6). Amplicon library preparation and semiconductor sequencing were done according to the manufacturers’ instructions using the Ion AmpliSeq Kit for Chef DL8, the Ion 510 & Ion 520 & Ion 530 Kit—Chef, the Ion 530 Chip Kit on the Ion Chef, and the Ion GeneStudio S5 prime system (Thermo Fisher Scientific). Output files were generated with Torrent Suite 5.12.0. Variant calling of non-synonymous somatic variants compared to the human reference sequence was performed using the Ion Reporter Software (Thermo Fisher Scientific, Version 5.16.0.2). Variants called by the Ion Reporter Software were visualized using the Integrative Genomics Viewer (IGV 2.8.0)^{75 (link)} to exclude panel-specific artifacts.

Frontzek F., Staiger A.M., Zapukhlyak M., Xu W., Bonzheim I., Borgmann V., Sander P., Baptista M.J., Heming J.N., Berning P., Wullenkord R., Erdmann T., Lutz M., Veratti P., Ehrenfeld S., Wienand K., Horn H., Goodlad J.R., Wilson M.R., Anagnostopoulos I., Lamping M., Gonzalez-Barca E., Climent F., Salar A., Castellvi J., Abrisqueta P., Menarguez J., Aldamiz T., Richter J., Klapper W., Tzankov A., Dirnhofer S., Rosenwald A., Mate J.L., Tapia G., Lenz P., Miething C., Hartmann W., Chapuy B., Fend F., Ott G., Navarro J.T., Grau M, & Lenz G. (2021). Molecular and functional profiling identifies therapeutically targetable vulnerabilities in plasmablastic lymphoma. Nature Communications, 12, 5183.

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SARS-CoV-2 Genome Sequencing Protocol

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Samples were randomly selected from the 1508 specimens that underwent HRM screening. 479 barcoded libraries were prepared using the Ion AmpliSeq SARS-CoV-2 Research Panel (Thermo Fisher Scientific) and the Ion AmpliSeq Library Kit Plus (Thermo Fisher Scientific), following manufacturer’s protocol. The panel consists of 237 amplicons that allows for the sequencing of 99% of the SARS-CoV-2 reference genome (NC_045512.2), covering from position 43 to position 29,842. All the reactions were performed in a Veriti™Dx 96-Well Thermal Cycler (Applied Biosystems™). Libraries were quantified with the Qubit dsDNA HS Assay kit (Life Technologies) and then diluted to 30 pM. Libraries were loaded into the Ion Chef™ Instrument (Thermo Fisher Scientific) for template enrichment and chip loading. Sequencing was performed with the Ion S5 GeneStudio Sequencer using the Ion 510 & Ion 520 & Ion 530 Kit-Chef and the Ion 530™ chip-kit (all Thermo Fisher Scientific).

Mio C., Dal Secco C., Marzinotto S., Bruno C., Pimpo S., Betto E., Bertoni M., Pipan C., Sozio E., Tascini C., Damante G, & Curcio F. (2021). Local occurrence and fast spread of B.1.1.7 lineage: A glimpse into Friuli Venezia Giulia. PLoS ONE, 16(12), e0261229.

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High-Throughput BRCA Mutation Profiling

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Libraries were prepared using the Oncomine BRCA Research panel (Thermo Fisher Scientific, Waltham, MA, USA) following to the manufacturer's instructions. In brief, barcoded libraries were generated using 10 ng of DNA from each sample using an Ion AmpliSeq™ Library Kit Plus (Thermo Fisher Scientific) and an Oncomine™ BRCA research assay (Thermo Fisher Scientific). Two premixed pools of 265 primer pairs were used to generate sequencing libraries. Clonal amplification of libraries was carried out by emulsion polymerase chain reaction using an Ion Chef system (Thermo Fisher Scientific). The resulting libraries were sequenced on an Ion S5 plus Sequencer using an Ion 520 Chip and the Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef (Thermo Fisher Scientific).

Chao A., Lin Y.H., Yang L.Y., Wu R.C., Chang W.Y., Chang P.Y., Chang S.C., Lin C.Y., Huang H.J., Lin C.T., Chou H.H., Huang K.G., Kuo W.L., Chang T.C, & Lai C.H. (2019). BRCA1/2 mutation status in patients with metachronous breast and ovarian malignancies: clues towards the implementation of genetic counseling. Journal of Gynecologic Oncology, 31(3), e24.

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Targeted Myeloid Disorder Mutation Analysis

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Targeted mutation analyses were performed by Next Generation Sequencing (NGS; Ion GeneStudio S5 prime, Thermo Fisher Scientific, Waltham, MA, USA) using an AmpliSeq custom panel designed for myeloid disorders comprising hotspot regions in 21 genes (JAK2, FLT3, STAT3, ASXL1, IDH1, IDH2, SRSF2, SF3B1, U2AF1, SETBP1, MPL, KIT, CBL, CSF3R, CALR, ETNK1, KRAS, NRAS, HRAS, BRAF, GNAS) and the 10 genes (CEBPA, RUNX1, IKZF1, DNMT3A, EZH2, ZRSR2, TP53, TET2, NPM1, STAG2). Amplicon library preparation and semiconductor sequencing was done according to the manufacturers’ manuals using the Ion AmpliSeq Library Kit v2.0, the Ion Library TaqMan Quantitation Kit, the Ion 510 & Ion 520 & Ion 530 Kit—Chef and the Ion 520 Chip Kit (Thermo Fisher Scientific, Fermont, CA, USA). Variant calling of non-synonymous somatic variants compared to the human reference sequence was performed using Ion Reporter Software (Thermo Fisher Scientific, Version 5.12.3.0). Variants were filtered with a threshold allele frequency of 5%.
Variants called by the Ion Reporter Software were visualized using the Integrative Genomics Viewer (IGV; Broad Institute, Cambridge, MA, USA; Version 2.5.2) to exclude panel-specific artefacts.

Bauer M., Vaxevanis C., Al-Ali H.K., Jaekel N., Naumann C.L., Schaffrath J., Rau A., Seliger B, & Wickenhauser C. (2021). Altered Spatial Composition of the Immune Cell Repertoire in Association to CD34+ Blasts in Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemia. Cancers, 13(2), 186.

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Targeted NF1 Gene Sequencing Panel

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A targeted sequencing NGS custom panel was designed for the NF1 gene using the Ion AmpliSeq Designer tool available from Thermo Fisher Scientific, Carlsbad, CA (https://www. ampliseq.com/). The panel contained 234 amplicons covering 21.87 kb of the genome including the entire coding region of the NF1 gene. Ten nanograms of DNA obtained from tumoral tissue and blood were used to prepare libraries using the Ion AmpliSeq Kit for Chef DL8 (Thermo Fisher Scientific, Carlsbad, CA) on the Ion Chef System (Thermo Fisher Scientific, Carlsbad, CA). Libraries were pooled together, and the Ion 510 & Ion 520 & Ion 530 Kit-Chef (Thermo Fisher Scientific, Carlsbad, CA) was used in the Ion Chef System for template preparation, followed by sequencing on the Ion S5 System using the Ion 520 Chip Kit (Thermo Fisher Scientific, Carlsbad, CA).
Raw data have been processed automatically on the Torrent Server and aligned to the reference hg19 genome. Further analysis was performed by the Ion Reporter Analysis Server v. 5.18 (Thermo Fisher Scientific, Carlsbad, CA) for variant calling and annotation using a custom bioinformatic workflow for "Tumor vs Normal" to filter out germline SNP from the analysis.

Ciampi R., Ramone T., Romei C., Casalini R., Matrone A., Prete A., Gambale C., Minardi S.P., Caparezza G., Pierotti M.A., Torregrossa L., Ugolini C., Materazzi G, & Elisei R. (2023). NF1 Gene Inactivation Acts as Tumor Driver in RET/RAS Negative Medullary Thyroid Carcinomas. European journal of endocrinology.

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