Ion ampliseq cancer panel

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion AmpliSeq Cancer Panel is a targeted next-generation sequencing (NGS) panel designed to detect mutations in a set of genes associated with various types of cancer. The panel is intended to enable researchers to interrogate specific genomic regions of interest related to cancer-associated genes.

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12 protocols using ion ampliseq cancer panel

Ion Torrent-based Cancer Gene Panel

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Twenty ng of DNA were used for multiplex PCR amplification using the Ion AmpliSeq Cancer Panel (Life Technologies) that explores selected regions of the following 46 cancer-associated genes: ABL1, ALK, AKT1, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR/VEGFR2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RET, RB1, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL.
The quality of the obtained library was evaluated by the Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies). Emulsion PCR was performed either manually or with the OneTouch DL system (Life Technologies). Sequencing was run on the Ion Torrent Personal Genome Machine (PGM, Life Technologies) loaded with a 316 chip as per manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome as well as variant calling and filtering, was done using the Torrent Suite Software v.3.6 (Life Technologies) with default options for the Ion AmpliSeq Cancer Panel. Variants were annotated using the SnpEff software v.3.4 [12] (link) and the NCBI mRNA Reference Sequences listed in Table S2. Alignments were visually verified with the Integrative Genomics Viewer; IGV v.2.3 [13] (link).

Mafficini A., Amato E., Fassan M., Simbolo M., Antonello D., Vicentini C., Scardoni M., Bersani S., Gottardi M., Rusev B., Malpeli G., Corbo V., Barbi S., Sikora K.O., Lawlor R.T., Tortora G, & Scarpa A. (2014). Reporting Tumor Molecular Heterogeneity in Histopathological Diagnosis. PLoS ONE, 9(8), e104979.

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Targeted NGS of Bladder Cancer

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A number of 22 bladder cancer samples, the same ones analyzed by microarray (except one samples from microarray patien cohort with low DNA concentration), were sequenced using Ion Ampliseq Cancer Panel and Ion Torrent PGM Next Generation Sequencing (Thermo Fischer Scientific); this panel contains the most relevant hot spot mutation. The amplicon libraries were prepared with 20 ng of DNA and the Ion Ampliseq™ Library Kit 2.0 (Life Technologies) and this was followed by a purification step using AMpure XP Beads (Beckman Coulter). Lastly, Qubit 2.0 was used for the quantification using Qubit HS DNA kit. For sequencing, four bar-coded 100pM-diluted libraries were used for each Ion 316 Chip (Thermo Fischer Scientific). Ion Torrent PGM Machine (Thermo Fischer Scientific) performed the sequencing, using the Ion PGM HI-Q Sequencing 200 kit. The software Torrent Suit 5.6 and Ion Reporter 5.6 executed the bioinformatics analysis, specifically for data trimming alignment and variant calling.

Braicu C., Buiga R., Cojocneanu R., Buse M., Raduly L., Pop L.A., Chira S., Budisan L., Jurj A., Ciocan C., Magdo L., Irimie A., Dobrota F., Petrut B, & Berindan-Neagoe I. (2019). Connecting the dots between different networks: miRNAs associated with bladder cancer risk and progression. Journal of Experimental & Clinical Cancer Research : CR, 38, 433.

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Comprehensive Molecular Profiling Assays

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All testing was performed in a laboratory accredited by the College of American Pathologists (CAP) and certified to meet Clinical Laboratory Improvement Amendments (CLIA). Three molecular profiling assays were used over the study period: a custom multiplex genotyping panel on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass-spectrometry platform (MassARRAY, Agena Bioscience, San Diego, CA, USA) to genotype 279 mutations within 23 genes (Additional file 1: Table S1); the TruSeq Amplicon Cancer Panel (TSACP, Illumina) on the MiSeq sequencer (Illumina) covering regions of 48 genes (Additional file 1: Table S2); and the Ion AmpliSeq Cancer Panel (ASCP, ThermoFisher Scientific) on the Ion Proton sequencer (ThermoFisher Scientific) covering regions of 50 genes (Additional file 1: Table S3). For more in-depth methodology on molecular profiling assays, including sequence alignment and base calling, see Additional file 1: Supplementary Methods.

Stockley T.L., Oza A.M., Berman H.K., Leighl N.B., Knox J.J., Shepherd F.A., Chen E.X., Krzyzanowska M.K., Dhani N., Joshua A.M., Tsao M.S., Serra S., Clarke B., Roehrl M.H., Zhang T., Sukhai M.A., Califaretti N., Trinkaus M., Shaw P., van der Kwast T., Wang L., Virtanen C., Kim R.H., Razak A.R., Hansen A.R., Yu C., Pugh T.J., Kamel-Reid S., Siu L.L, & Bedard P.L. (2016). Molecular profiling of advanced solid tumors and patient outcomes with genotype-matched clinical trials: the Princess Margaret IMPACT/COMPACT trial. Genome Medicine, 8, 109.

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Comprehensive Genetic Profiling of Tumor DNA

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DNA from each tumor was extracted using a Qiagen DNA mini kit (Qiagen). A genomic DNA library was constructed from an Ion AmpliSeq Cancer Panel (49 genes for 190 regions) on Ion PGM (Thermo Fisher Scientific) according to the manufacturer’s protocol. The list of target genes is available at http://www.lifetechnologies.com/order/catalog/product/4471262. Single nucleotide variants were assessed using Torrent Variant Caller (version 4) with a genome viewer and igv software.

Ishida K., Ito C., Ohmori Y., Kume K., Sato K.A., Koizumi Y., Konta A., Iwaya T., Nukatsuka M., Kobunai T., Takechi T, & Nishizuka S.S. (2017). Inhibition of PI3K suppresses propagation of drug-tolerant cancer cell subpopulations enriched by 5-fluorouracil. Scientific Reports, 7, 2262.

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Ion AmpliSeq Cancer Panel Library Preparation

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The Ion AmpliSeq Cancer Panel (Life Technologies, Carlsbad, CA, USA) was used to generate target amplicon libraries, as described previously [23] (link). In brief, 10 ng of DNA was amplified by PCR using pre-mixed Ion AmpliSeq Cancer Primer Pools containing 190 primer pairs and the AmpliSeq HiFi Master Mix (Ion AmpliSeq Library Kit, Life Technologies). The 190 multiplexed amplicons were treated with FuPa Reagent (Life Technologies) for partial digestion of the primer sequences and phosphorylation. The amplicons were then ligated to adapters from the Ion Xpress Barcode Adapters 1–16 Kit (Life Technologies) according to the manufacturer's instructions. After ligation, the amplicons underwent nick-translation and additional library amplification by PCR to complete the linkage between the adapters and amplicons. The BioAnalyser High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA) was used to visualise the size and range and to determine the library concentrations.

Takano S., Fukasawa M., Maekawa S., Kadokura M., Miura M., Shindo H., Takahashi E., Sato T, & Enomoto N. (2014). Deep Sequencing of Cancer-Related Genes Revealed GNAS Mutations to Be Associated with Intraductal Papillary Mucinous Neoplasms and Its Main Pancreatic Duct Dilation. PLoS ONE, 9(6), e98718.

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Ion AmpliSeq Cancer Panel Sequencing

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The Ion Ampliseq Cancer Panel (Life Technologies) covering mutation hotspot regions of 46 genes was used for library preparation and sequencing using the IT-PGM sequencer (Life Technologies) as described previously (Singh et al, 2013 (link)). Briefly, starting from 10 ng FFPE DNA, library preparation was performed using an Ion Ampliseq 2.0 kit and IT Ampliseq Cancer Panel primers (Life Technologies). From the barcoded library, manual emulsion PCR was performed, followed by sequencing eight multiplexed samples using Ion 318 chips and the IT-PGM Torrent Suite v2.0 (Life Technologies) was used for analysis.

Singh R.R., Patel K.P., Routbort M.J., Aldape K., Lu X., Manekia J., Abraham R., Reddy N.G., Barkoh B.A., Veliyathu J., Medeiros L.J, & Luthra R. (2014). Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours. British Journal of Cancer, 111(10), 2014-2023.

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Comprehensive Tumor Genomic Profiling

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Mutational status of the parental tumor performed as a standard of care under CLIA environment was collected and included mutation status of KRAS, BRAF, and PIK3CA genes. Tumor cellularity was determined by hematoxylin and eosin staining of sections adjacent to those used for DNA extraction. Standard laboratory procedures were used to isolate sectioned tissue and then extract, purify, and quantify DNA. The gene analysis was performed as a standard of care at UT-MDACC as previously described (34 (link)). Ion Ampliseq Cancer Panel (Life Technologies, Grand Island, NY) was used to identify hotspot mutations found in 46 genes, which was expanded to 50 genes by adding the following genes EZH2, IDH2, GNA11, and GNAQ (34 (link)). Sequence reading, alignment and base calling were conducted using Torrent Suite software V2.0.1 (Life Technologies) with reference standards consisting of Human Genome Build 19 (Hg19). Genomic variant detection was performed using Torrent Variant Caller software V1.0 (Life Technologies)

Katsiampoura A., Raghav K., Jiang Z.Q., Menter D.G., Varkaris A., Morelli M.P., Manuel S., Wu J., Sorokin A.V., Rizi B.S., Bristow C., Tian F., Airhart S., Cheng M., Broom B.M., Morris J., Overman M.J., Powis G, & Kopetz S. (2017). Modeling of Patient Derived Xenografts in Colorectal Cancer. Molecular cancer therapeutics, 16(7), 1435-1442.

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Comprehensive Cancer Profiling of Tissue and Blood

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Tissue NGS was performed using multiple CLIA-compliant platforms: Ion Ampliseq Cancer Panel (Life Technologies, Grand Island, NY) (CMS 46/50), Oncomine Comprehensive Cancer Panel assay (ThermoFisher, Waltham, MA), Ion AmpliSeq Comprehensive Cancer Panels (ThermoFisher) (CMS400) performed at MDACC Molecular Diagnostic Laboratory, FoundationOne, MSK-IMPACT.^{24 (link)–28 (link)} Blood NGS was performed using Guardant360 (Guardant Health, Redwood City, CA) circulating cell-free DNA (cfDNA) testing.^{29 (link)} For the comparative exploratory analyses with CRC, sidedness and CMS profiles were extracted using CRC patients reported previously by MDACC, MSK and includes those from TCGA.^{22 (link)–24 (link)} The genetic profile was extracted using disclosed data from these CLIA tests for the patients.

Raghav K., Shen J.P., Jácome A.A., Guerra J.L., Scally C.P., Taggart M.W., Foo W.C., Matamoros A., Shaw K.R., Fournier K., Overman M.J, & Eng C. (2020). Integrated clinico-molecular profiling of appendiceal adenocarcinoma reveals a unique grade-driven entity distinct from colorectal cancer. British Journal of Cancer, 123(8), 1262-1270.

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Ion Torrent Amplicon Sequencing of Tumors

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Ion torrent deep amplicon sequencing of tumor samples: deoxyribonucleic acid was extracted from tumor tissues and quantitated using the Qubit2 fluorometer (Invitrogen, Grand Island, NY). Ten nanograms of DNA was used for multiplex PCR of a panel covering 739 mutations in 46 cancer-related genes (Ion AmpliSeq Cancer Panel, Life Technologies, Grand Island, NY). Subsequent processing of samples was performed according to the manufacturer's protocol. Library constructions of the amplicons and subsequent enrichment of the sequencing beads was performed using the OneTouch (Grand Island, NY) system. Sequencing was done on the 314 chip with 10 megabases capacity using the Ion Torrent Personal Genome Machine (Life Technologies) as per the manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome and base calling, was done using built-in software.

Saulnier Sholler G.L., Bond J.P., Bergendahl G., Dutta A., Dragon J., Neville K., Ferguson W., Roberts W., Eslin D., Kraveka J., Kaplan J., Mitchell D., Parikh N., Merchant M., Ashikaga T., Hanna G., Lescault P.J., Siniard A., Corneveaux J., Huentelman M, & Trent J. (2015). Feasibility of implementing molecular-guided therapy for the treatment of patients with relapsed or refractory neuroblastoma. Cancer Medicine, 4(6), 871-886.

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Somatic Hotspot Mutation Analysis in FFPE Tumors

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For somatic hotspot mutation analysis, DNA was extracted, purified, and quantified from microdissected, paraffin-embedded tumor specimens. Next generation sequencing for hotspot mutations was performed using the Ion Ampliseq Cancer Panel (Life Technologies, Grand Island, NY) in a Clinical Laboratory Improvement Amendments-certified Molecular Diagnostics Laboratory at MD Anderson [19 (link), 33 (link)]. A panel of 46 genes was initially tested and then expanded to 50 genes, as described previously [34 (link)].

Wang Z., Piha-Paul S., Janku F., Subbiah V., Shi N., Gong J., Wathoo C., Shaw K., Hess K., Broaddus R., Naing A., Hong D., Tsimberidou A.M., Karp D., Yao J., Meric-Bernstam F, & Fu S. (2017). Antiangiogenesis and gene aberration-related therapy may improve overall survival in patients with concurrent KRAS and TP53 hotspot mutant cancer. Oncotarget, 8(20), 33796-33806.

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