Amplicons. Deep sequencing was performed using the Ion Torrent platform (Life Technologies). Briefly, 10 ng of purified genomic DNA were used for library construction with the Ion AmpliSeq Colon and Lung Cancer Panel v2 (Life Technologies) that targets 504 mutational hotspot regions of the following 22 cancer-associated genes, in alphabetical order: AKT1, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, KRAS, MAP2K1, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, STK11, TP53. Emulsion PCR was performed either manually or with the OneTouch DL system (Life Technologies). The quality of the obtained library was evaluated by the Agilent® 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies; Santa Clara, CA). 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 and variant calling, was done using the Torrent Suite Software v.3.2 (Life Technologies). Filtered variants were annotated using both the Ion Reporter software v1.2 (Life Technologies) and the SnpEff software v.3.0 (alignments visually verified with the Integrative Genomics Viewer; IGV v.2.1, Broad Institute) [46 (link)].
Ion ampliseq colon and lung cancer panel v2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion AmpliSeq Colon and Lung Cancer Panel v2 is a targeted next-generation sequencing (NGS) panel designed to analyze genetic variants in colon and lung cancer-related genes. The panel includes a set of pre-designed, multiplexed primer pools that enable the amplification and sequencing of specific regions of interest within the genome.
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Lab products found in correlation
Ion torrent personal genome machine, by Thermo Fisher Scientific (2 mentions)
Ion ampliseq library kit 2, by Thermo Fisher Scientific (2 mentions)
Torrent suite software v3, by Thermo Fisher Scientific (1 mentions)
Ion torrent platform, by Thermo Fisher Scientific (1 mentions)
Onetouch dl system, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer on chip electrophoresis, by Agilent Technologies (1 mentions)
Therascreen kras pyro kit, by Qiagen (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Ion library taqman quantitation kit, by Thermo Fisher Scientific (1 mentions)
Ion chef instrument, by Thermo Fisher Scientific (1 mentions)
Ion torrent system, by Thermo Fisher Scientific (1 mentions)
Ion 318 v2 chip, by Thermo Fisher Scientific (1 mentions)
Ion personal genome machine hi q sequencing kit, by Thermo Fisher Scientific (1 mentions)
Ion 314 chip, by Thermo Fisher Scientific (1 mentions)
Ampure magnetic beads, by Beckman Coulter (1 mentions)
Ionxpress, by Thermo Fisher Scientific (1 mentions)
6 protocols using ion ampliseq colon and lung cancer panel v2
1
Ion Torrent Deep Sequencing of Cancer Genes
2
Tumor and cfDNA NGS Mutation Analysis
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Tumor and cfDNA samples were analyzed with NGS panels for mutation detection. For library preparation, tumor DNA (10 ng) and cfDNA (maximum of 3000 copies) were subjected to multiplex PCR amplification with the use of an Ion AmpliSeq Library Kit 2.0 (Life Technologies, Carlsbad, CA, USA) and Ion AmpliSeq Colon and Lung Cancer Panel v2 (Life Technologies), the latter of which targets 22 cancer‐associated genes, with 92 amplicons covering 1205 hotspot mutations. Details are provided in Doc. S1 .
3
Molecular Profiling of Tumor Samples
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Tumor tissue was available for all patients and the KRAS mutation status was assessed from the primary tumor (all patients except DR4) or metastatic sites (DR4). In addition, the primary tumors from patient DR7, DR9, and DR13 were examined for changes in TP53 and/or BRAF. The Ion AmpliSeq Colon and Lung Cancer Panel v2 (Thermo Fisher Scientific), the Therascreen KRAS Pyro Kit (QIAGEN) or the Idylla KRAS and NRAS-BRAF-EGFR S492R Mutation Assays (Biocartis) were used to establish the mutation status in these genes directly from formalin-fixed paraffin-embedded (FFPE) tissue sections. Tumor genotyping was carried out at the Diagnostic and Research Institute of Pathology, Medical University of Graz and at the Department of Pathology, General Hospital Graz II, Graz, Austria.
For 9 of 11 patients (82%), specific mutations were available from the primary tissue, in one patient (DR4) only metastatic material was available for molecular profiling and in the tumor of one patient (DR13), no mutation was identified.
For 9 of 11 patients (82%), specific mutations were available from the primary tissue, in one patient (DR4) only metastatic material was available for molecular profiling and in the tumor of one patient (DR13), no mutation was identified.
4
Comprehensive Genomic Profiling in Oncology
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Patients were assessed with NGS for mutational status on DNA from FFPE blocks or cytological smears using the Ion AmpliSeq™ Colon and Lung Cancer Panel v2 from Thermo Fisher Scientific, Waltham, MA, USA, as a part of the diagnostic workup process at the Department of Clinical Pathology at Sahlgrenska University Hospital, Gothenburg, Sweden, assessing hotspot mutations in EGFR, BRAF, KRAS and NRAS. Until June 2017, ALK fusions were assessed with immunohistochemistry (IHC), and with fluorescence in situ hybridization (FISH) if positive or inconclusive IHC; ROS1 was analyzed upon request with FISH. Thereafter, ALK, ROS1 and RET fusions were assessed on RNA using the Oncomine Solid Tumor Fusion Panel from Thermo Fisher Scientific.
5
Targeted DNA Sequencing of Colorectal Cancers
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After microscopic identification of areas with highest tumor cell concentration, DNA was isolated using a commercial DNA Extraction Kit (DNeasy; Qiagen) following the manufacturer’s protocol.
In cohort 1, multiplex PCR-based amplicon high-throughput sequencing was performed using the Ion Torrent system (ThermoFisher Scientific) according to the manufacturer’s recommendations. Briefly, 10 ng of genomic DNA was used for library construction with the Ion AmpliSeq Colon and Lung Cancer Panel v2 and the Ion AmpliSeq Library Kit 2.0 (ThermoFisher). Library quantification was carried out on the StepOne Plus Real-Time PCR System employing the Ion Library TaqMan Quantitation Kit (both ThermoFisher). Emulsion PCR and subsequent enrichment was done on the Ion Chef instrument (ThermoFisher). Data analysis was performed using the Sequence Pilot software (JSI medical systems GmbH). Mean sequencing depth for CTNNB1 exon 3 and commonly heterozygous CRC mutations (KRAS, PIK3CA, FBXW7) was 1816 (range 569–4241) and 1308 (range 482–3671), respectively.
CRCs from cohort 2 were analysed with more extended customized panels as previously reported [17 (link)]. Mean sequencing depth for CTNNB1 exon 3 and commonly heterozygous CRC mutations (KRAS, PIK3CA, FBXW7) was 6144 (range 1891–11,334) and 3473 (range 1342–9467), respectively.
In cohort 1, multiplex PCR-based amplicon high-throughput sequencing was performed using the Ion Torrent system (ThermoFisher Scientific) according to the manufacturer’s recommendations. Briefly, 10 ng of genomic DNA was used for library construction with the Ion AmpliSeq Colon and Lung Cancer Panel v2 and the Ion AmpliSeq Library Kit 2.0 (ThermoFisher). Library quantification was carried out on the StepOne Plus Real-Time PCR System employing the Ion Library TaqMan Quantitation Kit (both ThermoFisher). Emulsion PCR and subsequent enrichment was done on the Ion Chef instrument (ThermoFisher). Data analysis was performed using the Sequence Pilot software (JSI medical systems GmbH). Mean sequencing depth for CTNNB1 exon 3 and commonly heterozygous CRC mutations (KRAS, PIK3CA, FBXW7) was 1816 (range 569–4241) and 1308 (range 482–3671), respectively.
CRCs from cohort 2 were analysed with more extended customized panels as previously reported [17 (link)]. Mean sequencing depth for CTNNB1 exon 3 and commonly heterozygous CRC mutations (KRAS, PIK3CA, FBXW7) was 6144 (range 1891–11,334) and 3473 (range 1342–9467), respectively.
6
Targeted Sequencing of Colon and Lung Cancer Panels
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The Ion Ampliseq Colon and Lung Cancer Panel V2 and RNA-Fusion Panel (Thermo Fisher Scientific) were used to generate a library of amplicons for each sample with 10 ng of genomic DNA or complimentary DNA, respectively, using polymerase chain reaction (PCR) according to manufacturer's instructions without modification. The resulting DNA amplicons were partially digested with FuPa reagents and ligated to Ion Xpress (Thermo Fisher Scientific) adapters, and then purified using AMPure magnetic beads (Beckman Coulter, Brea, CA).
Emulsion PCR and enrichment were done using an Ion Chef instrument with Ion Personal Genome Machine (PGM) Hi-Q Sequencing kit (Thermo Fisher Scientific). Samples were then loaded onto an Ion 318 Chip v2 or Ion 314 Chip for sequencing by an IonTorrent Personal Genome Machine (PGM; Thermo Fisher Scientific) following manufacturer's instructions without modification. Mutations or rearrangements were detected in greater than 50 genes, including ALK, BRAF, EGFR, FGFR1, KIT, KRAS, MET, NRAS, PIK3CA, PTEN, RET, and TP53.
Emulsion PCR and enrichment were done using an Ion Chef instrument with Ion Personal Genome Machine (PGM) Hi-Q Sequencing kit (Thermo Fisher Scientific). Samples were then loaded onto an Ion 318 Chip v2 or Ion 314 Chip for sequencing by an IonTorrent Personal Genome Machine (PGM; Thermo Fisher Scientific) following manufacturer's instructions without modification. Mutations or rearrangements were detected in greater than 50 genes, including ALK, BRAF, EGFR, FGFR1, KIT, KRAS, MET, NRAS, PIK3CA, PTEN, RET, and TP53.
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