Ion ampliseq comprehensive cancer panel

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The Ion AmpliSeq Comprehensive Cancer Panel is a targeted next-generation sequencing (NGS) panel designed to analyze 409 cancer-related genes. The panel enables the detection of single nucleotide variants, insertions, deletions, and copy number variations across a wide range of cancer types.

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49 protocols using ion ampliseq comprehensive cancer panel

Comprehensive Cancer Gene Profiling

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For the primary tumor tissues, mutations in the 409 cancer-related genes were analyzed using an Ion AmpliSeq Library Kit 2.0 and Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) (Thermo Fisher Scientific, Waltham, MA, USA). Tumor DNA was extracted using All Prep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) and 40 ng of DNA were subjected to multiplex PCR amplification. Then, the Ion Xpress Barcode Adapters (Thermo Fisher Scientific) were ligated to the PCR products, which were then purified with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purified libraries were pooled, and then sequenced with the Ion Torrent S5 instrument and Ion 550 Chip Kit (both from Thermo Fisher Scientific). DNA sequencing data were accessed through the Torrent Suite v.5.10 program (Thermo Fisher Scientific). Reads were aligned against the hg19 human reference genome, and variants were called using Variant Call Format ver. 5.10. Raw variant calls were filtered by quality score <100, depth coverage <19, and were manually checked using the integrative genomics viewer (IGV; Broad Institute, Cambridge, MA, USA). Germline mutations were excluded using the Human Genetic Variation Database (http://www.genome.med.kyoto-u.ac.jp/SnpDB) and the Exome Aggregation Consortium database.

Ohara S., Suda K., Sakai K., Nishino M., Chiba M., Shimoji M., Takemoto T., Fujino T., Koga T., Hamada A., Soh J., Nishio K, & Mitsudomi T. (2020). Prognostic implications of preoperative versus postoperative circulating tumor DNA in surgically resected lung cancer patients: a pilot study. Translational Lung Cancer Research, 9(5), 1915-1923.

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Comprehensive Cancer Profiling of FFPE Biopsies

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Formalin-fixed paraffin-embedded (FFPE) macro-dissected specimens obtained during routine diagnostic biopsy and/or resection of primary tumor were used. Tumor cell percentage was evaluated by two pathologists (GP and AV) using hematoxylin/eosin-stained slides from adjacent sections. For all patients excluding those attaining a pathological complete response (pCR), i.e., absence of invasive cancer in breast and node surgical specimens, both pre- and post-NAC tumor samples were sequenced and matched germline DNA was obtained from negative lymph nodes or breast normal tissue. DNA was extracted using GeneRead DNA FFPE kit (Quiagen, Hilden, Germany) and analyzed using the IonAmpliseq™ Comprehensive Cancer Panel (CCP) (ThermoFisher, Waltham, MA, USA), which cover all exons of 409 cancer-related genes. Raw sequencing data were mapped to the human reference genome (hg19) using the TMAP algorithm implemented in the Torrent Suite software version 4.4.3 with default parameters. Aligned bam files for matched tumor and normal samples were analyzed for copy number alteration (CNAs) calling using ad-hoc workflow implemented on Ion Reporter version 5.10.

Silvestri M., Dugo M., Vismara M., De Cecco L., Lanzoni D., Vingiani A., Folli S., De Santis M.C., de Braud F., Pruneri G., Di Cosimo S, & Cappelletti V. (2022). Copy number alterations analysis of primary tumor tissue and circulating tumor cells from patients with early-stage triple negative breast cancer. Scientific Reports, 12, 1470.

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Targeted NGS for Liquid Biopsy

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The library for NGS evaluating ctDNA was constructed using the Oncomine Pan-Cancer Cell-Free Assay for 52 genes (Table S8), following the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, USA), using 4.4–22 ng of cfTNA and 29–31 ng of genomic DNA of buffy coat.
For tumor tissue, normal tissue, and buffy coat, libraries were prepared using 40–80 ng of extracted genomic DNA using the Ion Ampliseq Comprehensive Cancer Panel (CCP) (Thermo Fisher Scientific, Waltham, MA, USA) for 409 genes (Table S9), including genes frequently mutated in pancreatic cancer and Ion Xpress Barcode Adapters (Thermo Fisher Scientific, Waltham, USA) following the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA from the buffy coat was fragmented to 150 bp with S220 Focused ultrasonicator (Covaris, Woburn, MA, USA) before library preparation. The quality of all constructed libraries was evaluated using the High Sensitivity D1000 ScreenTape (Agilent, Santa Clara, CA, USA).

Watanabe K., Nakamura T., Kimura Y., Motoya M., Kojima S., Kuraya T., Murakami T., Kaneko T., Shinohara Y., Kitayama Y., Fukuda K., Hatanaka K.C., Mitsuhashi T., Pittella-Silva F., Yamaguchi T., Hirano S., Nakamura Y, & Low S.K. (2022). Tumor-Informed Approach Improved ctDNA Detection Rate in Resected Pancreatic Cancer. International Journal of Molecular Sciences, 23(19), 11521.

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Comprehensive Genomic Profiling Using Multiple Sequencing Platforms

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For whole-exome sequencing, high-quality genomic DNA (1.5 μg per sample) was subjected to capture using a SureSelect Human All Exon v5 + UTR kit according to the manufacturer’s protocol (Agilent Technologies). The qualified exome-captured libraries were sequenced using a HiSeq 2000 with the TruSeq PE Cluster kit v3 and TruSeq SBS kit v3 (all from Illumina) according to the manufacturer’s protocol. For RNA sequencing, libraries were prepared using the TruSeq RNA Access Library Prep Guide (Part # 15049525 Rev. B; Illumina) according to the manufacturer’s instructions. Equal concentrations of each library were sequenced using a NextSeq 500 (Illumina) platform. For target sequencing, libraries were prepared from each sample using an Ion AmpliSeq Comprehensive Cancer Panel (CCP; Thermo Fisher Scientific) following the manufacturer’s instructions. Equal concentrations of each library were sequenced using an Ion Proton System (Thermo Fisher Scientific).

Chen T.W., Lee C.C., Liu H., Wu C.S., Pickering C.R., Huang P.J., Wang J., Chang I.Y., Yeh Y.M., Chen C.D., Li H.P., Luo J.D., Tan B.C., Chan T.E., Hsueh C., Chu L.J., Chen Y.T., Zhang B., Yang C.Y., Wu C.C., Hsu C.W., See L.C., Tang P., Yu J.S., Liao W.C., Chiang W.F., Rodriguez H., Myers J.N., Chang K.P, & Chang Y.S. (2017). APOBEC3A is an oral cancer prognostic biomarker in Taiwanese carriers of an APOBEC deletion polymorphism. Nature Communications, 8, 465.

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Somatic Variant Profiling for Cancer

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The number of somatic variants detected on NGS, using the Ion AmpliSeq Comprehensive Cancer Panel (Thermo Fisher Scientific, Waltham, MA, USA), interrogated 1.29 Mb of the genome. We calculated the MB in variants per megabase (Mb) using maftools^{43 (link)}. Based on the ROC analysis to determine the MB cutoff point to predict MGC developments, we divided the MB levels into one of two groups: low (less than 7.75 variants/Mb) and high (≥ 7.75 variants/Mb).

Sakuta K., Sasaki Y., Abe Y., Sato H., Shoji M., Yaoita T., Yagi M., Mizumoto N., Onozato Y., Kon T., Koseki A., Sato S., Murakami R., Miyano Y, & Ueno Y. (2020). Somatic alterations and mutational burden are potential predictive factors for metachronous development of early gastric cancer. Scientific Reports, 10, 22071.

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Comprehensive Cancer Gene Sequencing

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Target sequencing for major tumor-related genes was conducted using the Ion AmpliSeq™ Comprehensive Cancer Panel (Thermo Fisher Scientific, 4477685), which covers all exons of 409 genes. The HBB gene was analyzed using a custom panel that included 5 amplicons designed by the AmpliSeq Designer. Gene expressions were examined using the Oncomine™ Myeloid Research Assay (Thermo Fisher Scientific, A36941) and the Ion AmpliSeq™ Transcriptome Human Gene Expression Panel (Thermo Fisher Scientific, A31446). Sequence libraries and templates were prepared using the Ion AmpliSeq Kit for Chef DL8 (Thermo Fisher Scientific, A29024), followed by the Ion 510™ & Ion 520™ & Ion 530™ Kit-Chef or Ion 540™ Kit-Chef (Thermo Fisher Scientific, A34461/A30011). Sequencing was performed on the Ion GeneStudio S5 System using the Ion 530 or 540 chip (Thermo Fisher Scientific, A27763/A27765). Reads were aligned to the hg19 reference or the hg19 AmpliSeq Transcriptome ERCC v1 reference. The sequence variants and fusion genes were analyzed using the Ion Reporter 5.20 (Thermo Fisher Scientific). The expression analysis was performed using the Transcriptome Analysis Console 4.0.2 (Thermo Fisher Scientific). Variant analysis of the sequence data was performed with the settings as shown in Table S1.

Kasai F., Mizukoshi K, & Nakamura Y. (2024). Variable characteristics overlooked in human K-562 leukemia cell lines with a common signature. Scientific Reports, 14, 9619.

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Comprehensive Cancer Panel Sequencing

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DNA samples from #3cHCC-CCA and #3N were subjected to amplicon sequencing using Ion AmpliSeq Comprehensive Cancer Panel (Thermo Fisher Scientific, Inc.), as described previously (13 (link)). Candidates for cancer (#3cHCC-CCA)-specific single-nucleotide variants (SNVs) were identified by Tumor-Normal Pair Analysis version 5.2 of Ion Reporter (https://ionreporter.thermofisher.com/ir/).

Ohni S., Yamaguchi H., Hirotani Y., Nakanishi Y., Midorikawa Y., Sugitani M., Naruse H., Nakayama T., Makishima M, & Esumi M. (2021). Direct molecular evidence for both multicentric and monoclonal carcinogenesis followed by transdifferentiation from hepatocellular carcinoma to cholangiocarcinoma in a case of metachronous liver cancer. Oncology Letters, 23(1), 22.

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Comprehensive Cancer Panel Sequencing

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DNA (40 ng) was used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel, enabling the targeted coverage of all exons of 409 cancer-related genes (Thermo Fisher Scientific). The 15,992 amplicons obtained represented more than 1.69 megabases of target sequence. Library preparation and sequencing with the Ion Torrent sequencer were performed as previously described (10 (link)). Somatic mutations (single nucleotide mutations, insertions, and deletions) and copy number variations (CNVs) were detected using statistical approaches in tumor and normal samples from the Ion Reporter software 5.0 tumor-normal workflow (Thermo Fisher Scientific). Recurrent genomic regions with CNVs were identified using copy numbers greater than 3 and less than 1 for gains and losses, respectively. Germline DNA mutations were identified using the Torrent Suite variant caller plugin 5.0 with high-stringency germline parameters and annotated using Ion Reporter.

Adachi Y., Mita H., Sasaki Y., Himori R., Onodera K., Nakamura M., Kikuchi T., Yamashita K., Yoshida Y., Ishii Y, & Endo T. (2018). Malignant paraganglioma of the posterior mediastinum: A case report with genetic analysis. Molecular and Clinical Oncology, 10(1), 10-16.

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Comprehensive Cancer Panel Mutation Profiling

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The Comprehensive Cancer Panel™ (CCP, IonAmpliSeq™ Comprehensive Cancer Panel™, Thermo Fisher Scientific™) was used to identify target mutations in exonic regions of 409 cancer-related genes. Four libraries were created using 40 ng from both FFPE DNA from diagnostic biopsies and germline DNA samples as per manufacturer's specification [17 (link)] Libraries were quality-checked on an Agilent™ TapeStation. The Thermo Fisher Scientific™ Ion Chef™ system was used for template preparation followed by sequencing on an Ion PGM™ System using Ion 318™ chips, one library per chip for FFPE DNA samples and four germline libraries per chip [18 (link)].

Cirmena G., Garuti A., De Mariano M., Coco S., Ferrando L., Isnaldi E., Barbero V., Fregatti P., Del Mastro L., Ferrando F., Gonella R., Garlaschi A., Friedman D., Ballestrero A, & Zoppoli G. (2020). Circulating Tumor DNA Using Tagged Targeted Deep Sequencing to Assess Minimal Residual Disease in Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy. Journal of Oncology, 2020, 8132507.

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Comprehensive Cancer Mutation Analysis

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Matched primary/metastatic lesions for a total of 13 samples (five primaries and eight metastases) were sequenced using the Ion Ampliseq Comprehensive Cancer Panel (Thermo Fisher Scientific), which targets the relevant regions of 409 genes. The complete gene list of this assay and details of the targeted regions can be found at http://www.thermofisher.com. Data analysis of HCTS and orthogonal validation of mutations were performed as described in supplementary material, Supplementary materials and methods.

Amato E., Mafficini A., Hirabayashi K., Lawlor R.T., Fassan M., Vicentini C., Barbi S., Delfino P., Sikora K., Rusev B., Simbolo M., Esposito I., Antonello D., Pea A., Sereni E., Ballotta M., Maggino L., Marchegiani G., Ohike N., Wood L.D., Salvia R., Klöppel G., Zamboni G., Scarpa A, & Corbo V. (2018). Molecular alterations associated with metastases of solid pseudopapillary neoplasms of the pancreas. The Journal of Pathology, 247(1), 123-134.

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