Ion onetouch enrichment system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion OneTouch Enrichment System is a laboratory instrument designed for the purification and enrichment of nucleic acid samples, such as DNA or RNA, in preparation for downstream analysis. The system utilizes magnetic bead-based technology to capture and purify target molecules from complex sample matrices. The core function of the Ion OneTouch Enrichment System is to provide a automated, streamlined workflow for nucleic acid sample preparation prior to further processing or analysis.

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9 protocols using ion onetouch enrichment system

Ion Torrent Library Preparation

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Purified amplicons were subjected to 2 rounds of size selection using 0.6x volumes of SPRI size select beads (Beckman Coulter) and eluted in 15 uL of water. Concentrations were measured using the Qubit HS dsDNA kit (Thermo Fisher Scientific). IgG1/2a/2b amplicons (amplified with IgG2b-AdA reverse primer) and IgG3 amplicons (amplified with IgG3-AdA reverse primer) were mixed 1:1, and the mix was in turn mixed in equimolar amounts with the kappa chain amplicons. Concentrations were adjusted to 50 pM and the mix was used to template IonSphere Particles using the Ion PGM Template IA 500 Kit (Thermo Fisher Scientific) following manufacturer's instructions. Templated particles were enriched using an Ion OneTouch Enrichment System (Thermo Fisher Scientific).

Farfán-Arribas D.J., Liu S., Wang S, & Lu S. (2017). The dynamics of immunoglobulin V-gene usage and clonotype expansion in mice after prime and boost immunizations as analyzed by NGS. Human Vaccines & Immunotherapeutics, 13(12), 2987-2995.

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Ion Torrent-based Transcriptome and Genotyping

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Emulsion PCR (emPCR) was performed with the Ion PGM Template IA 500 Kit (Thermo Fisher Scientific) and GeneAmp PCR system 9700 (Thermo Fisher Scientific) for transcription analysis of FLA-I genes and with the Ion 520 and 530 Kit-OT2 and OneTouch 2 instrument (Thermo Fisher Scientific) for genotyping of FLA-I and FLA-DRB genes and confirmation of genotyping data in FLA-E/H/K genes. After the emulsion PCR, the beads carrying the single-stranded DNA templates were enriched with the Ion OneTouch Enrichment System (Thermo Fisher Scientific) according to the manufacture’s recommendation. Sequencing was performed using the Ion PGM Hi-Q View Sequencing Kit and Ion 316 Chip Kit (Thermo Fisher Scientific) for transcription analysis and using the Ion S5 Sequencing Kit and Ion 520/530 Chip Kit (Thermo Fisher Scientific) for genotyping of FLA-I and FLA-DRB genes and confirmation of genotyping data in FLA-E/H/K genes.
The raw data processing and base-calling, trimming and output of quality-filter sequence reads that were binned on the basis of the Ion Xpress Barcodes into separate sequence fastq files, were all performed by the Torrent Suite 4.2.1 (Thermo Fisher Scientific) for the transcription analysis and by the Torrent Suite 5.6.0 (Thermo Fisher Scientific) for the genotyping with full processing for shotgun analysis.

Okano M., Miyamae J., Suzuki S., Nishiya K., Katakura F., Kulski J.K., Moritomo T, & Shiina T. (2020). Identification of Novel Alleles and Structural Haplotypes of Major Histocompatibility Complex Class I and DRB Genes in Domestic Cat (Felis catus) by a Newly Developed NGS-Based Genotyping Method. Frontiers in Genetics, 11, 750.

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Ion Torrent PGM Sequencing Protocol

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Pooled, barcoded libraries were clonally amplified using the Ion OneTouch™ 2 instrument from the Ion PGM™ Template OT2 200 kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions (20 (link)). Ion sphere particles were enriched using the Ion One Touch™ enrichment system (Thermo Fisher Scientific, Inc). The subsequently enriched template-positive ion sphere particles were loaded onto an Ion 316™ chip and sequenced on the PGM using the Ion PGM sequencing 200 kit v2 (Thermo Fisher Scientific, Inc.). Data from sequencing runs on the Ion Torrent PGM™ were automatically transferred to the Torrent Server hosting the Torrent Suite™ Software v4.4.3 (Thermo Fisher Scientific, Inc.). The Torrent Suite Software uses the Torrent Browser, which includes the Torrent Mapping Alignment Program and Torrent Variant Caller for alignment and variant detection. A human genome reference sequence (hg19) was used as a reference. Ion Reporter™ Software (Thermo Fisher Scientific, Inc.) was used to perform variant calling and mapping. A >2% mutation frequency was defined as positive.

Kameta E., Sugimori K., Kaneko T., Ishii T., Miwa H., Sato T., Ishii Y., Sue S., Sasaki T., Yamashita Y., Shibata W., Matsumoto N, & Maeda S. (2016). Diagnosis of pancreatic lesions collected by endoscopic ultrasound-guided fine-needle aspiration using next-generation sequencing. Oncology Letters, 12(5), 3875-3881.

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Ion Sphere Particle Enrichment Protocol

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Clonal amplification of the libraries was carried out by emulsion PCR performed by using the Ion OneTouch 200 Template Kit v2 (Cat. No. A29900, Thermo Fisher Scientific) on the Ion OneTouch™ System (Thermo Fisher Scientific) according to the manufacturer's instructions. Subsequently, enrichment for template-positive Ion Sphere™ Particles was achieved using the Ion PGM™ Enrichment Beads (Cat no. 4478525, Thermo Fisher Scientific) on the Ion OneTouch Enrichment System (Thermo Fisher Scientific), following the protocol recommended by manufacturers.

Tavano F., Gioffreda D., Fontana A., Palmieri O., Gentile A., Latiano T., Latiano A., Latiano T.P., Scaramuzzi M., Maiello E., Bazzocchi F, & Perri F. (2023). Evaluation of inherited germline mutations in cancer susceptibility genes among pancreatic cancer patients: a single-center study. Molecular Medicine, 29, 14.

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Ion Torrent PGM Sequencing Protocol

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Libraries were normalized and pooled to an equimolar concentration as recommended by the manufacturer. The pooled libraries was used to generate template-positive Ion Sphere Particles containing clonally amplified DNA. Emulsion PCR was performed on the Ion OneTouch 2 instrument with the Ion PGM Template OT2 200 Kit according the template preparation protocol provided by Ion Torrent (Thermo Fisher). Template-positive Ion Sphere Particles were enriched using the Ion OneTouch Enrichment System and subsequently loaded onto Ion 318 v2 Chips (Thermo Fisher). Sequencing was performed using the Ion PGM System with the Ion PGM Sequencing 200 Kit v2 following the recommended protocol (Thermo Fisher).

Ma K., Li H., Cao Y., Zhao X., Liu W, & Zhao X. (2016). Haplotype diversity in mitochondrial genome in a Chinese Han population. Journal of human genetics, 61(10).

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Ion Sphere™ Particle Preparation

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Using emulsion PCR, the generated libraries are attached to beads and further amplified. The concentration of each of the libraries was determined with qPCR using the Ion Library Quantitation Kit (Life Technologies, a part of Thermo Fisher Scientific Inc.), and the template dilution factors were calculated. High‐quality templated Ion Sphere™ particles, containing massively parallel clonally amplified DNA, were prepared for the 200 base‐read libraries using the Ion PGM™ Template OT2 200 Kit and the Ion OneTouch™ 2 System (Life Technologies, a part of Thermo Fisher Scientific Inc.). The template‐positive Ion Sphere™ particles were enriched with the Ion OneTouch™ Enrichment System (Life Technologies, a part of Thermo Fisher Scientific Inc.) as per the manufacturer's guidelines.

Chaitanya L., Ralf A., van Oven M., Kupiec T., Chang J., Lagacé R, & Kayser M. (2015). Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine. Human Mutation, 36(12), 1236-1247.

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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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Ion Torrent 16SV6 rDNA Sequencing

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Sequencing was preceded by clonal amplification, by an emulsion PCR, of the 16SV6 rDNA amplicons. To accomplish this, each of the four amplicons was diluted to a concentration of 1.56 × 10⁸ DNA molecules per microliter and then a pool was formed by mixing 5 µL of each of distinct amplicons. The pooled DNA was attached to the surface of ion sphere particles (ISPs) using the IonPGM™ Template OT2 400 kit (Life Technologies, cod 4480974) and the corresponding protocol (OT2 400 kit, Publication No. MAN0007219, Rev. 2.0). The emulsion PCR was carried out in the Ion OneTouch™ 2 System (Life Technologies), the quality of amplification was checked and ISP enrichment was performed in the Ion OneTouch™ Enrichment System (Life Technologies). Sequencing primers were then annealed to the ISPs’ single stranded DNA, following Ion PGM sequencing 400 kit protocol (Life Technologies Publication No. MAN0007242). Sequencing proceeded in the Ion torrent personal genome machine (PGM™, Life Technologies) in a ISP loaded 314™ chip. Low quality and polyclonal sequence reads, as well as primers and barcodes were filtered out of the data and the 16SV6 rDNA sequences were available as a FastQ file.

da Silva Santos A.C., Jensen J.R., de Oliveira S.L, & Rodrigues J. (2017). Gut dysbiosis in mice genetically selected for low antibody production. Gut Pathogens, 9, 43.

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Ion Torrent PGM Sequencing Protocol

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Template preparation was performed with the Ion PGM OneTouch OT2 200 Template Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. The library was diluted to a final concentration of 26 pM. 20 μL of the 20 code libraries above were mixed with equal volumes of amplification solution and emulsion PCR was performed using the Ion One Touch 2 instrument (Life Technologies, Carlsbad, CA, USA). An Ion OneTouch Enrichment System (Life Technologies, Carlsbad, CA, USA) was utilized to isolate template-positive Ion Sphere Particles, producing an enriched library with 3′ end beads.
The library was sequenced using the Personal Genome Machine (Ion Torrent PGM, Life Technologies, Carlsbad, CA, USA). The results were analyzed with the Ion Torrent PGM server system (Torrent Suite 4.2) which exported raw data along with a sequencing report.

Li L., Chen L., Li J., Zhang W., Liao Y., Chen J, & Sun Z. (2016). Correlational study on mitochondrial DNA mutations as potential risk factors in breast cancer. Oncotarget, 7(21), 31270-31283.

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