Genomic DNA of primary tumor, MUG Mel1 and clones was isolated on a Maxwell, MDxResearch System (Promega). Highly multiplexed PCR was used to generate amplicon libraries covering the coding region of 50 genes commonly implicated in cancer (Cancer Hotspot Panel v2, Cat.Nr. 4475346; Thermo Fisher Scientific, Waltham, MA). All analyses were performed in duplicates. Libraries were prepared using the Ion AmpliSeq Library Kit 2.0 and sequencing was performed on an Ion Proton Sequencer (Thermo Fisher Scientific). Emulsion PCR and sequencing runs were performed with the appropriate kits (Ion One Touch Template Kit version 2 and Ion Proton 200 Sequencing Kit; Thermo Fisher Scientific) using Ion PI chips. Sequencing length was set to 520 flows and yielded reads ranging from 70 to 150 bp, consistent with the expected amplicon size range. Initial data analysis was performed using the Ion Torrent Suite Software version 4.1 Plug-ins (Thermo Fisher Scientific)44 .
Ion proton 200 sequencing kit
Manufactured by Thermo Fisher Scientific
The Ion Proton 200 Sequencing Kit is a laboratory equipment product designed for DNA sequencing. It is part of the Ion Proton Sequencing System offered by Thermo Fisher Scientific.
Automatically generated - may contain errors
Lab products found in correlation
Ion proton sequencer, by Thermo Fisher Scientific (4 mentions)
Ion ampliseq library kit 2, by Thermo Fisher Scientific (4 mentions)
Ion one touch template kit version 2, by Thermo Fisher Scientific (4 mentions)
Ion onetouch 2 system, by Thermo Fisher Scientific (3 mentions)
Ion proton, by Thermo Fisher Scientific (3 mentions)
Ion pi template ot2 200 kit, by Thermo Fisher Scientific (2 mentions)
Torrent suite version 3, by Thermo Fisher Scientific (2 mentions)
Ion ampliseq rna fusion workflow, by Thermo Fisher Scientific (2 mentions)
Ion torrent suite software version 4.1 plug ins, by Thermo Fisher Scientific (1 mentions)
Ion pi template ot2 200 kit v3, by Thermo Fisher Scientific (1 mentions)
Ion onetouch es, by Thermo Fisher Scientific (1 mentions)
Cancer hotspot panel v2, by Thermo Fisher Scientific (1 mentions)
Trizol, by Merck Group (1 mentions)
Dynabeads mrna direct kit, by Thermo Fisher Scientific (1 mentions)
Ion total rna seq kit, by Thermo Fisher Scientific (1 mentions)
Ion reporter software, by Thermo Fisher Scientific (1 mentions)
Ion ampliseq exome rdy kit, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Qiaxcel advanced, by Qiagen (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
10 protocols using ion proton 200 sequencing kit
1
Targeted DNA Sequencing of Cancer Genes
2
Comprehensive RNA-Seq Data Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Libraries were clonally amplified by emulsion PCR on Ion Sphere Particles using Ion PI template OT2 200 Kit v3 (ThermoFisher) on an Ion OneTouch 2 system (ThermoFisher). Template-positive Ion Sphere Particles were enriched using an Ion OneTouch ES (ThermoFisher), and were processed for sequencing using an Ion Proton 200 Sequencing Kit and were loaded onto a P1 chip and sequenced with an Ion Proton (ThermoFisher) using default parameters (single-end, forward sequencing). Base calling, adaptor trimming, barcode deconvolution and alignment was performed on Torrent Suite version 3.6 (ThermoFisher) using the STAR RNA-seq aligner plugin. The number of reads per sample was 15–20 million, with an average read depth of 500 reads per gene. The mean read length for all samples was 124 bp with 97% accuracy overall. The Partek Genomic Suite 6.6 software (Partek Incorporated, St Louis, MO, USA) was used to analyse the data. Reads per kilobase per million normalisation for RNA-seq44 (link) was used using RefSeq transcript (2016-02-02) annotation followed by a one-way analysis of variance for gene differential expression. Gene targets showing a ∓2-fold change with P-values of <0.05 were put into DAVID, Metacore and IPA Ingenuity platforms for pathway and GO analysis.
3
Comprehensive Mutation Profiling of Oncogenes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Highly multiplexed PCR was used to generate amplicon libraries to amplify 207 amplicons, covering approximately 2800 COSMIC mutations from 50 oncogenes and tumor suppressor genes. (Cancer Hotspot Panel v2, Cat. Nr. 4475346; Thermo Fisher Scientific, Waltham, MA, USA). All analyses were performed in duplicate. Libraries were prepared using the Ion AmpliSeq Library Kit 2.0 and sequencing was performed on an Ion Proton Sequencer (Thermo Fisher Scientific, Waltham, MA, USA). Emulsion PCR and sequencing runs were performed with the appropriate kits (Ion One Touch Template Kit version 2 and Ion Proton 200 Sequencing Kit; Thermo Fisher Scientific) using Ion PI chips. Sequencing length was set to 520 flows and yielded reads ranging from 70 to 150 bp, consistent with the expected amplicon size range. Initial data analysis was performed using the Ion Torrent Suite Software version 4.1 Plug-ins (Thermo Fisher Scientific).
4
Cancer Hotspot Targeted Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA of MUG-Myx2a and MUG-Myx2b was isolated on a Maxwell, MDxResearch System (Promega). Highly multiplexed PCR was used to generate amplicon libraries covering the coding region of 50 genes commonly implicated in cancer (Cancer Hotspot Panel v2, Cat.Nr. 4475346; Thermo Fisher Scientific, Waltham, MA). All analyses were performed in duplicates. Libraries were prepared using the Ion AmpliSeq Library Kit 2.0 (Cat.Nr. 4475345; Thermo Fisher Scientific) and sequencing was performed on an Ion Proton Sequencer (Thermo Fisher Scientific). Emulsion PCR and sequencing runs were performed with the appropriate kits (Ion One Touch Template Kit version 2 and Ion Proton 200 Sequencing Kit; Thermo Fisher Scientific) using Ion PI chips. Sequencing length was set to 520 flows and yielded reads ranging from 70 to 150 bp, consistent with the expected amplicon size range.
5
Targeted Cancer Mutation Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For molecular analysis serial sections from 39 samples were obtained with first and last levels stained for hematoxylin and eosin to ensure optimal tissue selection. Unstained, intermediate sections were mounted on glass slides for selective manual microdissection with a scalpel. Genomic DNA was isolated on a Maxwell MDxResearch System (Promega). Using the Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA) 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes were amplified. Amplicon libraries were prepared using the Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific). Emulsion PCR and sequencing runs were performed with the appropriate kits (Ion One Touch Template Kit version 2 and Ion Proton 200 Sequencing Kit; Thermo Fisher Scientific) using Ion PI chips and sub-sequentially sequenced on an Ion Proton Sequencer (Thermo Fisher Scientific). Sequencing length was set to 520 flows and yielded reads ranging from 70 to 150 bp, consistent with the expected amplicon size range. For improved variant detection all samples were run in technical duplicates.
6
Comprehensive Transcriptome Profiling of Human Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from cells by using Trizol (Sigma, Gillingham, Dorest. UK). The reverse transcription to cDNA was performed with IonAmpliseqTM Transcriptome Human Gene Expression kit (Life Technologies). We then used the Human Gene Expression Core Panel primer set (Life Technologies) to prepare small amplicon gene expression libraries targeting 20,000 genes (95% of the RefSeq gene database). The cDNA amplicon libraries (125–300 bp) were ligated to adapters and amplified using IonXpress RNA-seq barcoded primers (5′). cDNA libraries were clonally amplified using an Ion PI template OT2 200 kit (Life technologies, USA) on an Ion OneTouch2 system (Life technologies) as per the manufacturer’s instructions. Samples were processed using the Ion Proton 200 sequencing kit and loaded onto a P1 chip and sequenced on an Ion Proton (Life technologies) using default parameters (single-end, forward sequencing). Base calling, adaptor trimming, barcode deconvolution, alignment and Ampliseq gene expression analysis was performed on Torrent Suite version 3.6 (Life technologies).
7
Ion Proton RNA-seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PolyA mRNA was isolated from total RNA using the Dynabeads mRNA DIRECT Kit (Life Technologies, UK). RNA fragment libraries (150–200 bp) were generated using the Ion Total RNA-Seq kit (Life Technologies), ligated to adaptors for cDNA synthesis and amplified using IonXpress RNA-seq barcoded primers (5′). cDNA libraries were clonally amplified using Ion PI template OT2 200 kit (Life technologies, USA) on an Ion OneTouch2 system (Life technologies) as per manufacturer’s instructions. Samples were processed using the Ion Proton 200 sequencing kit and loaded onto a P1 chip and sequenced on an Ion Proton (Life technologies) using default parameters (single-end, forward sequencing). Base calling, adaptor trimming, barcode deconvolution and alignment was performed on Torrent Suite version 3.6 (Life technologies) using the STAR RNA-seq aligner plugin. The Partek Genomic Suite 6.6 software was used for data analysis. The RPKM normalisation method for RNA-seq69 (link) was used followed by a 1-way Anova test for differential expression (from n = 4 samples per group).
8
Ion Torrent Exome Sequencing and Variant Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Ion AmpliSeq Exome RDY Kit (Thermo Fisher Scientific) was used to construct an Ion Torrent adapter-ligated library according to the manufacturer’s instructions. The Ion Proton Sequencing 200 Kit (Thermo Fisher Scientific) was used for nucleotide sequencing according to the manufacturer’s protocol. Germline and somatic mutations were detected using the torrent variant caller analysis and Ion Reporter software (Thermo Fisher Scientific), respectively. Detected mutations were validated using the Integrative Genomics Viewer (IGV),7 (link),8 (link) followed by Sanger and pyrosequencing. We searched COSMIC (http://cancer.sanger.ac.uk/cosmic/ ) and the Human Gene Mutation Database (HGMD (http://www.hgmd.cf.ac.uk/ac/index.php )) Professional v.2016.3 (BIOBASE, Beverly, MA, USA) to determine whether detected mutations had been previously reported as being associated with diseases. All of the genomic linear positions were based on human genome reference version UCSC Genome Assembly GRCh37.9 (link)
9
Ion Proton Fusion Gene Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fusion gene analysis was performed using the Ion Proton System, as previously reported53 (link). In brief, total RNA (10 ng) was used as a template to prepare cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). The Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific) and the Ion Proton Sequencing 200 Kit (Thermo Fisher Scientific) were used to construct an Ion Torrent adapter-ligated library and perform nucleotide sequencing, respectively, according to the manufacturer’s protocols. All data were analyzed using the Ion Reporter server. The Ion AmpliSeq RNA Fusion workflow (Thermo Fisher Scientific) was used to detect fusion transcripts from a panel of 491 fusion genes.
10
Detection of ERO1L-FERMT2 Fusion Transcript
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ethical statement. The Institutional Review Boards construct an Ion Torrent adapter-ligated library in accordance with the manufacturer's instructions, and the Ion Proton Sequencing 200 Kit (Thermo Fisher Scientific) was used for nucleotide sequencing in accordance with the manufacturer's protocol.
Detection of ERO1L-FERMT2 trasncript by RT-PCR. Total RNA of HCC1395 was used for the reverse transcription with the SuperScript III (Thermo Fisher Scientific), following the manufacturer's instructions. PCR reaction was performed by HotSter-Taq Master Mix (QIAGEN) with the forward primer 5'-TGAAGAGGCCGTGTCCTTTC-3' and reverse primer 5'-AGGGCTGCAAACATCATCATTT-3'. The amplification profile of the PCR consisted of 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. The PCR products were analysed by QIAxcel Advanced (QIAGEN) .
Data analysis. All data were analyzed using the Ion Reporter server. The Ion AmpliSeq RNA Fusion workflow (Thermo Fisher Scientific) was used to detect fusion transcripts targeted by the HOPE fusion panel. The reference file for this workflow was constructed from all fusion variants in the HOPE fusion gene panel. The workflow used a related BED file that describes the breakpoints between the two genes that are associated with each fusion. The reference and BED files are included in the Ion Reporter Software.
Detection of ERO1L-FERMT2 trasncript by RT-PCR. Total RNA of HCC1395 was used for the reverse transcription with the SuperScript III (Thermo Fisher Scientific), following the manufacturer's instructions. PCR reaction was performed by HotSter-Taq Master Mix (QIAGEN) with the forward primer 5'-TGAAGAGGCCGTGTCCTTTC-3' and reverse primer 5'-AGGGCTGCAAACATCATCATTT-3'. The amplification profile of the PCR consisted of 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. The PCR products were analysed by QIAxcel Advanced (QIAGEN) .
Data analysis. All data were analyzed using the Ion Reporter server. The Ion AmpliSeq RNA Fusion workflow (Thermo Fisher Scientific) was used to detect fusion transcripts targeted by the HOPE fusion panel. The reference file for this workflow was constructed from all fusion variants in the HOPE fusion gene panel. The workflow used a related BED file that describes the breakpoints between the two genes that are associated with each fusion. The reference and BED files are included in the Ion Reporter Software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!