318 chip

Manufactured by Thermo Fisher Scientific

Sourced in United States

The 318 chip is a laboratory equipment product designed for nucleic acid sequencing applications. It provides a platform for high-throughput DNA or RNA sequencing, enabling efficient and accurate genetic analysis.

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Ion 318 Chip Kit v2 (27 citations) Ion 318 Chip Kit (5 citations) Ion 318 Chip Kit v2 BC (4 citations) Ion 318TM chip (2 citations) Ion 318TM chip kit v2 (2 citations) Ion 318TM chip v2 (2 citations) 318 v2 Chips (2 citations) 1G-Ion 318 Chip Kit v2 (2 citations) Ion Torrent PGM 316 or 318 chip (2 citations) 318 Chip Kit v2 (2 citations)

13 protocols using 318 chip

Targeted NGS of Dystrophin Gene

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Targeted NGS covering the dystrophin gene was performed on genomic DNA extracted from blood lymphocytes of patients. Multiplex primer pools were designed using Ion AmpliSeq Designer software (Thermo Fisher Scientific, MA, USA). This custom gene panel covers 99.6% of the exonic region, including the flanking region (30 base pairs from the exon–intron boundary), and it covers 100% of the coding region. Enrichment of exonic sequences was performed with an Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific, MA, USA) and sequenced on an Ion PGM (Thermo Fisher Scientific, MA, USA) using 318 Chip (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol.

Okubo M., Minami N., Goto K., Goto Y., Noguchi S., Mitsuhashi S, & Nishino I. (2016). Genetic diagnosis of Duchenne/Becker muscular dystrophy using next-generation sequencing: validation analysis of DMD mutations. Journal of Human Genetics, 61(6), 483-489.

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Targeted Sequencing of Proteasome Subunits in Multiple Myeloma Cell Lines

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We performed DNA targeted sequencing in 8 multiple myeloma cell lines using semiconductor-sequencing technology (IonTorrent PGM) as per manufacturer’s protocol (25 (link)). All coding regions of the proteasome subunits were amplified in 200bp amplicons by multiplex PCR using customized oligos (Ion Ampliseq designer). Libraries were templated and enriched using IonOneTouch2 and IonOneTouch ES automated systems, respectively. Samples were sequenced using the 318™ chip (ThermoFisher). Raw data was aligned and indexed in BAM and BAI files using the IonTorrent suite. Variants were called using IonTorrent Somatic VariantCaller and low stringency settings (ThermoFisher). VCF files were annotated using a standalone application developed by the Mayo Clinic Bioinformatics Program called BioR (Biological Annotation Data Repository)(26 (link)). Somatic variants with a Mapping Quality <20, read depth <10X, or found in <1% of reads were removed. To remove germline mutations, common variants were eliminated based on the minor allele frequencies (>0.01%) available in one of the following germline variant databases: 1000 Genomes Project, ExAC and ESP6500, unless present in known CLL/MBL mutation hotspots or in COSMIC. Finally, variants of significant interest were visually inspected using Integrative Genomics Viewer (IGV)(27 (link)).

Shi C.X., Zhu Y.X., Bruins L.A., de Campos C.B., Stewart W., Braggio E, & Stewart A.K. (2020). Proteasome subunits differentially control Myeloma cell viability and proteasome inhibitor sensitivity. Molecular cancer research : MCR, 18(10), 1453-1464.

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Ion Torrent Sequencing of PCR Products

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Qubit 2.0 Fluorometer (Invitrogen, CA, USA) was used to quantify the purified PCR products. The amplicons were then evaluated for fragment concentration using Ion Library Quantitation Kit (Life Technologies, CA, USA). Concentrations were adjusted to 26 pM for all samples. Amplicons were attached to Ion Sphere Particles (ISPs) using an Ion PGM Template OT2 400 kit (Life Technologies, CA, USA) according to the manufacturer’s instructions. Multiplexed sequencing was conducted using 318 chip (Life Technologies, CA, USA) on the Ion Torrent Personal Genome platform (Life Technologies, CA, USA). Sequences were sorted by sample and filtered within the PGM software to remove low quality reads. Finally, the data from each sample was exported as individual FastQ files.

Gajardo K., Rodiles A., Kortner T.M., Krogdahl Å., Bakke A.M., Merrifield D.L, & Sørum H. (2016). A high-resolution map of the gut microbiota in Atlantic salmon (Salmo salar): A basis for comparative gut microbial research. Scientific Reports, 6, 30893.

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Targeted Sequencing of Tumor DNA

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Twenty nanograms of tumor DNA were used for the Ion Torrent library preparation of a panel covering 59 actionable genes (Supplementary Table 3) following the manufacturer's protocol for the Ion AmpliSeq Library Kit 2.0 (Life Technologies #4475345). The size distribution of the DNA amplicons was analyzed on the 2200 TapeStation (Agilent) using the High sensitivity kit (Agilent #5067-4626). Template preparation, emulsion PCR, and Ion Sphere Particle (ISP) enrichment was performed using the One Touch 2 kit (Life Technologies) according to manufacturer's instructions. The ISPs were loaded onto a 318 chip (Life Technologies #4484355) and sequenced using an Ion PGM 200 V2 sequencing kit (Life Technologies #4482006) on the Ion Torrent PGM for 500 cycles. The raw signal data were analyzed using NextGENe Software Suite v3.4.2 (Soft genetics).
The pipeline includes quality score assignment, alignment to the human genome 19 reference, mapping quality QC, coverage analysis and variant calling. After completion of the primary data analysis, lists of detected sequence variants (SNVs and INDELs) were compiled in the VCF (Variant Call File) format. For downstream analysis, variants with minimum coverage of 100 reads containing at least 10 of the mutant reads were selected. Variant calls were further analyzed using variant filtering and annotation using COSMIC v.64 and dbSNP build 135.

Jiang X., Pissaloux D., De La Fouchardiere C., Desseigne F., Wang Q., Attignon V., Fondrevelle M.E., De La Fouchardiere A., Perol M., Cassier P., Seigne C., Perol D., Coquard I.R., Meeus P., Fayette J., Flechon A., Le Cesne A., Penel N., Tredan O, & Blay J.Y. (2015). The sum of gains and losses of genes encoding the protein tyrosine kinase targets predicts response to multi-kinase inhibitor treatment: Characterization, validation, and prognostic value. Oncotarget, 6(28), 26388-26399.

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Intestinal Microbiome Profiling via 16S rRNA

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To analyze the microbial population of the distal intestinal digesta and mucosa, amplification of the variable regions V1 and V2 of the 16S rRNA was performed. The PCR was conducted using the universal bacterial primers 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′), 338R-I (5′-GCW GCC TCC CGT AGG AGT 3′), and 338R-II (5′-GCW GCC ACC CGT AGG TGT-3′) (57 (link)). The reactions were carried out in 50-μl volumes using 1 μl of DNA template, 25 μl of Phusion high-fidelity PCR master mix (Thermo Scientific, CA) and 1 μl of forward and reverse (pooled 338R-I and II) primers (5 μM). The PCR was run as follows: initial denaturation at 98°C for 3 min, followed by 35 cycles of denaturation at 98°C for 15 s, annealing decreasing from 63°C to 53°C in 10 cycles for 30 s, followed in turn by 25 cycles at 53°C for 30 s and an extension at 72°C for 30 s; followed by a final extension at 72°C for 10 min. PCR products were then analyzed in a 1.5% agarose gel and purified using the QIAquick PCR purification kit (Qiagen). High-throughput sequencing of the purified PCR products was carried out using the Ion Torrent Personal Genome Machine system (Life Technologies, California) as described elsewhere (26 (link)) using a 318 chip (Life Technologies). Obtained sequences were grouped by sample and filtered within an Ion Torrent Personal Genome Machine software to remove low-quality reads.

Gajardo K., Jaramillo-Torres A., Kortner T.M., Merrifield D.L., Tinsley J., Bakke A.M, & Krogdahl Å. (2017). Alternative Protein Sources in the Diet Modulate Microbiota and Functionality in the Distal Intestine of Atlantic Salmon (Salmo salar). Applied and Environmental Microbiology, 83(5), e02615-16.

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Whole-Genome Sequencing of Clinical Isolates

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A subset of the clinical isolates was analyzed using whole-genome sequencing (WGS). The bacterial genomes were sequenced using the IonTorrent PGM platform (Life Technologies, Carlsbad, USA) in accordance with the manufacturer’s instructions. The Ion Xpress Plus Fragment Library Kit was used to enzymatically shear 100 ng of the genomic DNA. The target fragment size was 400 bp. Subsequently, the fragmented DNA was processed using the Ion DNA Barcoding kit (Life Technologies), and its size was selected using the E-Gel SizeSelect 2% Agarose kit (Life Technologies). The size distribution of the DNA fragments was analyzed using the High Sensitivity kit (Agilent, Santa Clara, USA). Further sample processing was performed using the Ion OneTouch kit (Life Technologies). Finally, the amplified DNA was sequenced using the 318 chip (Life Technologies). The single reads obtained were de novo assembled using MIRA 3.9.9, which is a part of the Assembler plugin on the Ion Torrent server. The contigs were analyzed using the ResFinder version 4.1. web-service [80 (link)].

Mitteregger D., Wessely J., Barišić I., Bedenić B., Kosak D, & Kundi M. (2022). A Variant Carbapenem Inactivation Method (CIM) for Acinetobacter baumannii Group with Shortened Time-to-Result: rCIM-A. Pathogens, 11(4), 482.

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Ion Torrent Sequencing Protocol

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A Qubit Fluorimeter (Invitrogen, CA, USA) was used to quantify the purified PCR products. The amplicons were then evaluated for fragment concentration using Ion Library Quantitation Kit (Life Technologies, CA, USA). The concentration was adjusted to 26 pM. The amplicons were attached to ion sphere particles (ISPs) using the Ion PGM kit Template OT2 400 (Life Technologies, CA, USA) according to the manufacturer’s protocol. Multiplexed sequencing was conducted using the 318 chip (Life Technologies, CA, USA) on the Ion Torrent Personal Genome platform (Life Technologies, CA, USA). Sequences were sorted by sample and filtered within the PGM software to remove low-quality reads. Finally, the data for each sample were exported to an individual Fastq file.

Gainza O, & Romero J. (2020). Effect of mannan oligosaccharides on the microbiota and productivity parameters of Litopenaeus vannamei shrimp under intensive cultivation in Ecuador. Scientific Reports, 10, 2719.

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Whole Genome Sequencing of Bacterial Isolates

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Isolates were transported to the Molecular Diagnostics group at the Austrian Institute of Technology where they were checked for viability at arrival. Fifteen isolates were not viable after transportation, and sequencing failed in three isolates. After a viability check of all isolates, genomic DNA was extracted with the QiAmp DNA mini kit (Qiagen, Hilden, Germany). Whole genome sequencing (WGS) was performed using the Ion Torrent PGM platform using 400 bp read chemistry. Sequencing was performed according to the protocol recommended by Life Technologies. The Ion Xpress Plus Fragment Library Kit was used to enzymatically shear 100 ng of the genomic DNA. The target fragment size was 400 bp. Subsequently, the fragmented DNA was processed using the Ion DNA Barcoding kit (Life Technologies, Carlsbad, CA, USA) and its size selected using the E-Gel SizeSelect 2% Agarose kit (Life Technologies). The size distribution of the DNA fragments was analysed using the High Sensitivity Kit (Agilent, Santa Clara, Santa Clara, CA, USA). Further sample processing was performed using the Ion OneTouch Kit (Life Technologies). Finally, the amplified DNA was sequenced using the 318 chip (Life Technologies). Raw reads were assembled de novo using Assembler SPAdes software [7 (link)]. The genome was annotated using the RAST (Rapid Annotations using Subsystems Technology) database [8 (link),9 (link)].

D’Onofrio V., Cartuyvels R., Messiaen P.E., Barišić I, & Gyssens I.C. (2023). Virulence Factor Genes in Invasive Escherichia coli Are Associated with Clinical Outcomes and Disease Severity in Patients with Sepsis: A Prospective Observational Cohort Study. Microorganisms, 11(7), 1827.

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PIK3CA Mutation Detection Protocol

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Paraffin blocks were scraped in a selected area containing more than 50% of tumor cells, and DNA was extracted. Twenty nanograms of DNA was used for the Ion Torrent library preparation of exons 9 and 20 of PIK3CA gene (NM_006218.2) following the manufacturer’s protocol for the Ion AmpliSeq Libray Kit 2.0 (Life Technologies). The size distribution of the DNA amplicons was analyzed on the 2200 TapeSation (Agilent) using the high-sensitivity kit (Agilent). Template preparation, emulsion PCR, and ion sphere particle enrichment was performed using the One Touch 2 kit (Life Technologies) according to manufacturer’s instructions. The ion sphere particles were loaded onto a 318 chip (Life Technologies) and sequenced using an Ion PGM 200 v2 sequencing kit (Life Technologies) on the Ion Torrent PGM for 500 cycles. The raw signal data were analyzed using NextGENe Software Suite v3.4.2 (Softgenetics; NGS). The pipeline includes quality score assignment, alignment to human genome 19 reference, mapping quality QC, coverage analysis, and variant calling.

Azim H.A., Kassem L., Treilleux I., Wang Q., El Enein M.A., Anis S.E, & Bachelot T. (2016). Analysis of PI3K/mTOR Pathway Biomarkers and Their Prognostic Value in Women with Hormone Receptor–Positive, HER2-Negative Early Breast Cancer. Translational Oncology, 9(2), 114-123.

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Ion Amplicon Sequencing Protocol

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The Ion OneTouch Template Kit and the Ion OneTouch System (Life Technologies) were used to perform emulsion PCR and enrichment steps according to the manufacturer’s instructions. The 318 Chips (Life Technologies), Ion Xpress Barcode Adapters 1-16 Kit (Life Technologies), and Ion PGM System (Life Technologies) were then used to carry out the sequencing of amplicon libraries. After the sequencing was completed, the Torrent Mapping Alignment Program was used to map the readings of the reference genome (hg19). Sequencing success was determined by meeting a cut-off of 300,000 AQ20 readings on the 318V2 Chip. Torrent Variant Caller (3.6.6; Life Technologies) was then used to identify the variants. Minimum requirement of sequencing coverage is 500× to verify the credibility of variants.

Tian Y., Zhao X., Li P., Lu M, & Tian H. (2021). Correlation of EGFR G873R mutation with prognosis of docetaxel in non-small cell lung cancer. Journal of Thoracic Disease, 13(10), 5964-5972.

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