Ion 316v2 chips

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion 316v2 chip is a semiconductor-based sequencing chip designed for use with Ion Torrent sequencing systems. It is capable of generating high-quality DNA sequence data for a variety of applications, including targeted gene sequencing, small genome sequencing, and metagenomics analysis. The chip features a high-density array of semiconductor-based sensors that detect the release of hydrogen ions during the DNA sequencing process, allowing for rapid and efficient DNA sequencing.

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8 protocols using ion 316v2 chips

Mitochondrial DNA Sequencing Protocol

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PCR products were purified using QIAquick PCR purification kit (QIAGEN). Ion Torrent deep sequencing libraries were constructed from bisulfite-converted DNA using the KAPA Library Preparation Kit (Kapa Biosystems), quantified using the QIAxcel Advanced System (QIAGEN), and templated using the Ion PGM Template OT2 200 kit (Thermo Fisher). The libraries were sequenced using an Ion PGM Sequencing HiQ Kit with Ion 316 v2 Chips on the Ion Torrent PGM (Thermo Fisher), which generated non-directional ~200 nt length reads, 1500–7500 reads per library in the fastq format. Reads were aligned to human or mouse mtDNA reference sequences (hg38 and mm10, respectively) using the Bismark aligner [25 (link)] with bowtie2 option. Bisulfite conversion efficiency was evaluated based on cytosine conversion rate (C versus T) at non-CpG sites determined after visualization of the aligned reads (bam format) using the Integrative Genomics Viewer (IGV) [18 (link)].

Owa C., Poulin M., Yan L, & Shioda T. (2018). Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection. PLoS ONE, 13(2), e0192722.

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Targeted POLE and CTNNB1 Mutation Profiling

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Genomic DNA was extracted from 94 FFPE tissues, each containing at least 70% of tumor cells, starting from one 10 µm-thick section. DNA extraction was performed using the GeneRead DNA FFPE kit, according to manufacturer’s instructions (Qiagen, Hilden, Germany), and DNA samples were quantified with a Qubit fluorometer (ThermoFisher Scientific, Waltham, MA, USA).
Next-generation sequencing (NGS) was used to assess the mutational status of POLE (exons 9, 10, 11, 12, 13, 14, 19, 20, 26, 30, 36, 37, 39, 42, 46) and CTNNB1 (exon 3) with a custom AmpliSeq gene panel (Supplementary Materials, Table S1) on Ion 316 v2 chips (Thermo Fisher Scientific). Data analysis was performed using Ion Reporter v5.18.0.2 software (Thermo Fisher Scientific). Only patients with pathogenic variants in the POLE exonuclease domain have been included in the POLE mutant subgroup [23 (link)]. For more details on NGS analyses, see Appendix B.2. Quality control data of NGS output files for each patient, as well as cumulatively, in terms of total coverage, on-target sequencing, average depth and uniformity are shown in Supplementary Materials (Table S2, Figures S1 and S2).

Ravaggi A., Capoferri D., Ardighieri L., Ghini I., Ferrari F., Romani C., Bugatti M., Zanotti L., Vrede S., Tognon G., Pijnenborg J.M., Sartori E., Calza S., Bignotti E, & Odicino F. (2022). Integrated Biomarker Analysis Reveals L1CAM as a Potential Stratification Marker for No Specific Molecular Profile High-Risk Endometrial Carcinoma. Cancers, 14(21), 5429.

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Cancer Hotspot Panel Sequencing

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Next, all cases underwent next-generation sequencing based on Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHPv2) (Thermo Fisher Scientific) amplification using an Ion Torrent PGM platform (Thermo Fisher Scientific). This panel consists of 207 primer pairs interrogating 50 oncogenes and tumor suppressor genes (catalog no. 4475346, for panel details see AmpliSeq. com). LCM material was collected as described above and DNA extraction was performed using the Arcturus® PicoPure® DNA extraction kit (Applied Biosystems) according to the manufacturer's protocol for FFPE material with minor changes. The concentration was measured with a Qubit® 2.0 fluorometer using the Qubit® dsDNA HS Assay kit (Molecular Probes). CHPv2 libraries were constructed using 1-9.9 ng of DNA from each pooled laser-captured isolate. The samples were processed using the Ion AmpliSeq™ Library Kit 2.0 according to the recommended protocols and amplified by PCR for 20 or 23 cycles. The template and enrichment steps were carried out on the Ion OneTouch™ 2 System with Ion PGM™ Template OT2 200 Kit. The samples were adjusted to a concentration of 100 pM, applied to an Ion 316™ v2 Chips, and sequenced on an Ion PGM™ instrument (all kits and instruments from Thermo Fisher Scientific). The sequencing data were analyzed using the Ion Reporter plugin for Torrent Suite™ Software (v5.0.4).

Centeno I., Paasinen Sohns A., Flury M., Galván J.A., Zahnd S., Koelzer V.H., Sokol L., Dawson H.E., Lugli A., Cathomas G, & Zlobec I. (2017). DNA profiling of tumor buds in colorectal cancer indicates that they have the same mutation profile as the tumor from which they derive. Virchows Archiv : an international journal of pathology, 470(3).

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Ion PGM 200 Sequencing for Ampliseq Samples

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Ampliseq DesignSamples were subjected to the standard ion PGM 200 Sequencing v2 protocol using Ion 316 v2 chips (Life Technologies).

Di Matteo G., Chiriaco M., Scarselli A., Cifaldi C., Livadiotti S., Di Cesare S., Ferradini V., Aiuti A., Rossi P., Finocchi A, & Cancrini C. (2018). JAK3 mutations in Italian patients affected by SCID: New molecular aspects of a long‐known gene. Molecular Genetics & Genomic Medicine, 6(5), 713-721.

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Lymphoma Mutation Profiling Using NGS

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Genomic DNA was submitted to Next Generation Sequencing (NGS) using a laboratory-developed “Lymphopanel” set, designed to identify mutations in 34 genes important for lymphomagenesis (Supplementary Table 2). This design covers 87 703 bases and generates 872 amplicons. Amplified libraries were submitted to emulsion PCR with the Ion OneTouch™ 200 Template Kit (Life Technologies, California, USA) using the Ion OneTouch™ System (Life Technologies) according to the manufacturer's instructions. The generated Ion Sphere™ Particles (ISPs) were enriched with the Ion OneTouch™ Enrichment System and loaded and sequenced on Ion 316™ v2 Chips (Life Technologies).

Dubois S., Mareschal S., Picquenot J.M., Viailly P.J., Bohers E., Cornic M., Bertrand P., Veresezan E.L., Ruminy P., Maingonnat C., Marchand V., Lanic H., Penther D., Bastard C., Tilly H, & Jardin F. (2015). Immunohistochemical and genomic profiles of diffuse large B-cell lymphomas: Implications for targeted EZH2 inhibitor therapy?. Oncotarget, 6(18), 16712-16724.

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Phage Peptide Genome Amplification and Sequencing

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As previously described by Pleiko et al. [52] , peptideencoding region of bacteriophage genome was amplified by PCR using Phusion Green Hot Start II High-Fidelity DNA Polymerase (#F537L, Thermo Scientific) in 25 μL reaction volume. Cycling conditions: denaturation at 98 °C for 30 s, followed by 25 amplification cycles (10 s at 98 °C, 21 s at 72 °C), and final elongation (72 °C for 5 min). Polymerase chain reaction (PCR) products were purified using AMPure XP Bead Based Next-Generation Sequencing Cleanup system (Beckmann Coulter, A63881) using 1.8 μL of beads per 1 μL of PCR products. Purified PCR products were quantified using Agilent Bioanalyzer 2100 Instrument using the High sensitivity DNA Kit (#5067-4626, Agilent). Ion Torrent Emulsion PCR and enrichment steps were performed using Ion PGM HiQ View OT2 kit (#A29900, Life Technologies). High throughput sequencing (HTS) was performed using Ion Torrent™ Personal Genome Machine™ (ION-PGM) using Ion PGM HiQ View sequencing kit (#A30044, Life Technologies) and Ion 316v2 chips (#448,149, Life Technologies). The FASTQ sequence files were converted to text files and translated using in-house developed python scripts.

Tobi A., Haugas M., Rabi K., Sethi J., Põšnograjeva K., Paiste P., Jagomäe T., Pleiko K., Lingasamy P, & Teesalu T. (2024). Protease-activated CendR peptides targeting tenascin-C: mitigating off-target tissue accumulation. Drug delivery and translational research.

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High-Throughput Phage Genome Sequencing

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Peptide-encoding region of bacteriophage genome was amplified by PCR using Phusion Green Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific, F537L) (reaction volume: 25 μl, list of primers added as Supplementary Table S1). Cycling conditions: denaturation at 98°C for 30 s, followed by 25 amplification cycles (10 s at 98°C, 21 s at 72°C), and final elongation (72°C for 5 min). Polymerase chain reaction (PCR) products were purified using AMPure XP Bead Based Next-Generation Sequencing Cleanup system (Beckmann Coulter, A63881) using 1.8 μl of beads per 1 μl of PCR products. Purified PCR products were quantified using Agilent Bioanalyzer 2100 Instrument using the High sensitivity DNA Kit (Agilent, 5067-4626). Ion Torrent Emulsion PCR and enrichment steps were performed using Ion PGM HiQ View OT2 kit (Life technologies, A29900). HTS was performed using Ion Torrent™ Personal Genome Machine™ (ION-PGM) using Ion PGM HiQ View sequencing kit (Life technologies, A30044) and Ion 316v2 chips (Life Technologies, 448149). The FASTQ sequence files were converted to text files and translated using in-house developed python scripts.

Pleiko K., Põšnograjeva K., Haugas M., Paiste P., Tobi A., Kurm K., Riekstina U, & Teesalu T. (2021). In vivo phage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues. Nucleic Acids Research, 49(7), e38.

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Ion PGM Sequencing Protocol

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Sequencing was performed with the Ion PGM system using the Ion PGM ™ Sequencing 200 Kit v2 kit (Life Technologies, Massachusetts, USA). The Ion 316v2 chips (Life Technologies, Massachusetts, USA), with a capacity of 200-250 Mb, was chosen for this step.

Parco S., Benericetti G., Vascotto F, & Palmisciano G. (2019). Microbiome and diversity indices during blood stem cells transplantation - new perspectives?. Central European journal of public health, 27(4).

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