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Ion 520

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The Ion 520 is a laboratory instrument designed for DNA sequencing applications. It is capable of generating high-quality sequence data, but a detailed technical description cannot be provided in a concise and unbiased manner without the risk of making interpretations or extrapolations.

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Lab products found in correlation

Ion 530 kit chef, by Thermo Fisher Scientific (9 mentions) Ion 530 chip, by Thermo Fisher Scientific (5 mentions) Ion ampliseq library kit 2, by Thermo Fisher Scientific (4 mentions) Ion chef, by Thermo Fisher Scientific (3 mentions) Ion s5 system, by Thermo Fisher Scientific (3 mentions) Ion s5 sequencer, by Thermo Fisher Scientific (3 mentions) Ion s5 sequencing reagents, by Thermo Fisher Scientific (3 mentions) Ion chef system, by Thermo Fisher Scientific (3 mentions) Qubit fluorometer, by Thermo Fisher Scientific (3 mentions) Ion 510, by Thermo Fisher Scientific (2 mentions) Ion 530, by Thermo Fisher Scientific (2 mentions) Theoncomine lung cfdna assay, by Thermo Fisher Scientific (2 mentions) Ion xpress barcode adapter kit, by Thermo Fisher Scientific (2 mentions) Ion 530 chef reagents, by Thermo Fisher Scientific (2 mentions) Ion genestudio s5 system, by Thermo Fisher Scientific (2 mentions) Ion library taqman quantitation kit, by Thermo Fisher Scientific (2 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) Ion ampliseq comprehensive cancer panel, by Thermo Fisher Scientific (1 mentions) Dneasy powersoil pro kit, by Qiagen (1 mentions) Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)

17 protocols using ion 520

1

Targeted Cancer Gene Sequencing Protocol

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Library preparation was performed from 10 ng of DNA from each sample, which was added to the multiplex PCR reaction for library preparation using the Ion AmpliSeq Library Kit 2.0, Ion AmpliSeq Cancer Hotspot Panel v2 Kit (CHPv2), according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). CHPv2 contains 207 pairs of primers, covering hotspots in the following 50 genes: ABL1, EZH2, JAK3, PTEN, ACT1, FBXW7, IDH2, PTPN11, ALK, FGFR1, KDR, RB1, APC, FGFR2, KIT, RET, ATM, FGFR3, KRAS, SMAD4, BRAF, FLT3, MET, SMARCB1, CDH1, GNA11, MLH1, SMO, CDKN2A, GNAS, MPL, SRC, CSF1R, GNAQ, NOTCH1, STK11, CTNNB1, HNF1A, NPM1, TP53, EGFR, HRAS, NRAS, VHL, ERBB2, IDH1, PDGFR, ERBB4, JAK2 and PIK3CA. Clonally amplified templates for NGS were prepared using the Ion Chef System and Ion 520 and Ion 530 Kit-Chef according to the manufacturer’s instructions (Thermo Fisher Scientific). The obtained barcoded libraries were loaded onto four Ion 530 chips and sequenced using Ion 520 and Ion 530 Kit-Chef and Ion S5 System according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA).
Zięba S., Pouwer A.F., Kowalik A., Zalewski K., Rusetska N., Bakuła-Zalewska E., Kopczyński J., Pijnenborg J.M., de Hullu J.A, & Kowalewska M. (2020). Somatic Mutation Profiling in Premalignant Lesions of Vulvar Squamous Cell Carcinoma. International Journal of Molecular Sciences, 21(14), 4880.
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2

NGS-based IDH mutation detection

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The IDH mutation status of samples with unclear immunohistochemistry and of patients younger than 55 years with negative IDH immunohistochemistry was determined by NGS using the Ion AmpliSeq™ Comprehensive Cancer Panel (Life Technologies, Darmstadt, Germany). NGS libraries were generated with the Ion AmpliSeqTM Library 2.0 Kit (Life Technologies, Darmstadt, Germany, User guide MAN0006735 Rev. B.0). DNA was pretreated for 30 min with 1 unit Uracil-DNA Glycosylase (Life Technologies, Darmstadt, Germany). Template preparation, chip loading, and sequencing were performed with the Ion Chef™ and the Ion GeneStudio™ S5 Plus Systems according to the Ion 510™ & Ion 520™ & Ion 530™ Kit—Chef user guide (MAN0016854 Rev. E.0, Life Technologies, Darmstadt, Germany). Data were analyzed with Torrent Suite™ software version 5.12.2 and Ion Reporter software version 5.16. Read alignments at the IDH1 and IDH2 hotspots were visually inspected with the Integrative Genomics Viewer (Broad Institute, Cambridge, MA, USA).
Bumes E., Fellner C., Fellner F.A., Fleischanderl K., Häckl M., Lenz S., Linker R., Mirus T., Oefner P.J., Paar C., Proescholdt M.A., Riemenschneider M.J., Rosengarth K., Weis S., Wendl C., Wimmer S., Hau P., Gronwald W, & Hutterer M. (2022). Validation Study for Non-Invasive Prediction of IDH Mutation Status in Patients with Glioma Using In Vivo 1H-Magnetic Resonance Spectroscopy and Machine Learning. Cancers, 14(11), 2762.
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3

Ion Sequencing of Microbial Communities

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Multiplexed samples were prepared for sequencing employing the Ion 520 and Ion 530 Kit-Chef (Life Technologies, United States) according to the manufacturer’s instructions. Prepared samples were loaded on an Ion 530 Chip and then sequenced using an Ion GeneStudio S5 (Life Technologies, United States) at 850 reads per run. After sequencing, individual sequence reads were filtered by the PGM software to remove low quality and polyclonal sequences. Those reads were analyzed using QIIME (v1.9.1) (Caporaso et al., 2011 (link)), the analysis included OTU clustering, Alpha-diversity analysis, OTU analysis and species annotation. The OTU assigning method was UCLUST and the taxonomy assigning method was BLAST. The sequence similarity threshold for both OTU and taxonomy assignments was 97%. The taxonomy database employed was GreenGenes for 16s rRNA gene sequences. Principal component analysis (PCA) was used to compare reactors microbial communities. PCA was performed using Matlab.
Ribera-Pi J., Campitelli A., Badia-Fabregat M., Jubany I., Martínez-Lladó X., McAdam E., Jefferson B, & Soares A. (2020). Hydrolysis and Methanogenesis in UASB-AnMBR Treating Municipal Wastewater Under Psychrophilic Conditions: Importance of Reactor Configuration and Inoculum. Frontiers in Bioengineering and Biotechnology, 8, 567695.
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4

Microbial Profiling of Biomass

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Microbial composition of the biomass used for the study was analyzed. DNA extraction from a biomass sample was performed using the DNeasy PowerSoil Pro Kit (Qiagen, German) according to its principles and instructions and stored at −70°C. Two variable regions (V3, V4) of the 16S rRNA gene were amplified by polymerase chain reaction (PCR) with custom designed fusion primers described by Torrell et al. (2021 ). Bovine serum albumin (BSA) was added to the PCR reaction to neutralize potential inhibitors. The PCR product, called amplicon or library, was visualized with a 2% agarose gel, purified with the NucleoSpin kit (Macherey-Nagel, Berlin, Germany), and quantified with an Agilent 2100 Bioanalyzer (Agilent Technologies, California, USA) and the Agilent High Sensitivity DNA kit (Agilent Technologies). Lastly, an equimolar mixture (60pM) of the samples was created and the Ion 520 and Ion 530 Kit-Chef (Life Technologies, Carlsbad, California, USA) and a 530 chip were used for the sequencing on LifeTechnologies GeneStudio S5 machine, using 850 flows per run. The obtained data was analyzed using the software QIIME 2–2020.8.
Páez D.F., Villalba X.G., Zabalo N.A., Galceran H.T., Güell I.J, & Noguera X.G. (2023). Mass transfer vectors for nitric oxide removal through biological treatments. Environmental Science and Pollution Research International, 30(51), 110089-110103.
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5

SARS-CoV-2 Sequencing and Lineage Analysis

Cited 1 time
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Sequencing was performed on an Ion S5 semiconductor sequencer (Thermo Fisher Scientific, Waltham, MA, USA) using the Ion 510 and Ion 520 and Ion 530 kits according to the manufacturer’s protocol. Aligned files were obtained as BAM format from the TSS; these files were converted to FASTQ using the FileExporter plugin of TSS, and then FASTA files were used for downstream analysis. Phylogenetic lineages were assigned to genomic sequences by the SARS-CoV-2 Insight panel workflow and were double-checked using the PANGOLIN lineage tool v.4.0.1/PLEARN-v1.2.133 [46 (link)].
Ciubotariu I.I., Wilkes R.P., Kattoor J.J., Christian E.N., Carpi G, & Kitchen A. (2024). Investigating the rise of Omicron variant through genomic surveillance of SARS-CoV-2 infections in a highly vaccinated university population. Microbial Genomics, 10(2), 001194.
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6

Validating Multiplex PCR-NGS Assay for cfDNA Analysis

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Cross-platform validation was performed in 28 patients with M-Seq confirmed SNV(s)
within one or more hotspots targeted by a generic multiplex PCR-NGS panel (Extended Table 2a-b, Supplementary Table 8).
20ng of isolated cfDNA was used for library preparation using the
Oncomine™ Lung cfDNA Assay (ThermoFisher Scientific), according to the
manufacturer’s instructions. Automated template preparation and chip
loading was conducted on the Ion Chef™ instrument using the Ion
520™ & Ion 530™ Kit-Chef (ThermoFisher Scientific).
Ultimately, samples were sequenced on Ion 530™ chips using the Ion
S5™ System (ThermoFisher Scientific). Sequencing data was accessed on the
Torrent suite v5.2.2. Reads were aligned against the human genome (hg19) using
Alignment v4.0-r77189, and variants were called using the coverage Analysis
v4.0-r77897 plugin. All 18 bespoke-panel ctDNA negative patients had no tumor
SNVs detectable in plasma pre-operatively by the generic panel supporting
biological specificity of the bespoke targeted approach, 7 of 10 bespoke-panel
ctDNA positive patients had tumor SNVs detected in plasma by the generic panel
(Extended Table 2a-b). SNVs detected
by hotspot panel not identified by M-Seq are displayed in Extended Table 2c.
Abbosh C., Birkbak N.J., Wilson G.A., Jamal-Hanjani M., Constantin T., Salari R., Le Quesne J., Moore D.A., Veeriah S., Rosenthal R., Marafioti T., Kirkizlar E., Watkins T.B., McGranahan N., Ward S., Martinson L., Riley J., Fraioli F., Al Bakir M., Grönroos E., Zambrana F., Endozo R., Bi W.L., Fennessy F.M., Sponer N., Johnson D., Laycock J., Shafi S., Czyzewska-Khan J., Rowan A., Chambers T., Matthews N., Turajlic S., Hiley C., Lee S.M., Forster M.D., Ahmad T., Falzon M., Borg E., Lawrence D., Hayward M., Kolvekar S., Panagiotopoulos N., Janes S.M., Thakrar R., Ahmed A., Blackhall F., Summers Y., Hafez D., Naik A., Ganguly A., Kareht S., Shah R., Joseph L., Quinn A.M., Crosbie P., Naidu B., Middleton G., Langman G., Trotter S., Nicolson M., Remmen H., Kerr K., Chetty M., Gomersall L., Fennell D., Nakas A., Rathinam S., Anand G., Khan S., Russell P., Ezhil V., Ismail B., Irvin-sellers M., Prakash V., Lester J., Kornaszewska M., Attanoos R., Adams H., Davies H., Oukrif D., Akarca A.U., Hartley J.A., Lowe H.L., Lock S., Iles N., Bell H., Ngai Y., Elgar G., Szallasi Z., Schwarz R.F., Herrero J., Stewart A., Quezada S.A., Peggs K.S., Van Loo P., Dive C., Lin J., Rabinowitz M., Aerts H.J., Hackshaw A., Shaw J.A., Zimmermann B.G, & Swanton C. (2017). Phylogenetic ctDNA analysis depicts early stage lung cancer evolution. Nature, 545(7655), 446-451.
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7

Targeted Deep Sequencing of CRC Samples

Cited 2 times
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We performed target deep sequencing of the genomic DNA extracted from the three CRC tissue samples and matched normal samples using a custom NGS panel (OncoChase-AS01, ConnectaGen, Seoul, Korea), targeting 95 cancer-related genes as described elsewhere [10 (link),11 (link)]. Tumor DNA was amplified, digested, and barcoded using the Ion Ampliseq library kit 2.0 (Thermo Fisher Scientific) and Ion Xpress barcode adapter kit (Thermo Fisher Scientific) as described elsewhere [10 (link)]. The libraries were then templated on an Ion Chef system (Thermo Fisher Scientific) using Ion 520 and Ion 530 Chef reagents (Thermo Fisher Scientific). The prepared libraries were sequenced on an Ion S5 sequencer using an Ion 530 chip and Ion S5 sequencing reagents (Thermo Fisher Scientific) as described elsewhere [10 (link)].
Min S., Shin S, & Chung Y.J. (2019). Detection of KRAS mutations in plasma cell-free DNA of colorectal cancer patients and comparison with cancer panel data for tissue samples of the same cancers. Genomics & Informatics, 17(4), e42.
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8

SARS-CoV-2 Whole Genome Sequencing Protocol

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For whole viral genome sequencing, total RNA was reverse transcribed using Invitrogen SuperScript VILOTM cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). One hundred and seven samples were analyzed in eight different sequencing runs using the Ion Torrent S5 system (Thermo Fisher Scientific, Waltham, MA, USA) after library preparation, consisting of fragmentation and adapter ligation onto the PCR products and clonal amplification. cDNA libraries were then prepared using the Ion AmpliSeq SARS-CoV-2 Research Panel (Thermo Fisher Scientific, Waltham, MA, USA). After quantification of cDNA libraries with Real-Time Step One PCR System (Thermo Fisher Scientific, Waltham, MA, USA), the prepared samples of ion sphere particles (ISP) were loaded onto an Ion 520™ chip with the Ion Chef (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed using the Ion S5™ sequencing reagents (Thermo Fisher Scientific, Waltham, MA, USA). The Torrent Suite 5.14.0 platform and specific plugins were used for NGS data analysis. All analysed sequences showed an alignment accuracy of over 96% and a base coverage over 20× (Figure 1). The pangolin software was used for the assignment of SARS-CoV-2 lineages. All sequences were then submitted as FASTA files on gisaid.org, which provides open access to genomic data on SARS-CoV-2.
Anaclerio F., Ferrante R., Mandatori D., Antonucci I., Capanna M., Damiani V., Tomo P.D., Ferrante R., Ranaudo M., De Laurenzi V., Stuppia L, & De Fabritiis S. (2021). Different Strategies for the Identification of SARS-CoV-2 Variants in the Laboratory Practice. Genes, 12(9), 1428.
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9

Ion Torrent Sequencing of Barcoded Libraries

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Libraries were normalized by dilution to 50 pM and either 16 or 32 libraries were pooled equimolarly. The clonal amplification of the barcoded DNA library (AmpliSeq libraries) onto ion spheres was performed using the Ion Chef™ instrument using Ion 520™ & Ion 530™ Kit-Chef according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The obtained barcoded libraries were multiplexed and loaded onto Ion 530™ Chips following the manufacturer’s protocol, and sequencing was executed on the Ion Gene Studio S5 system (Thermo Fisher Scientific, Waltham, MA, USA).
Smok-Kalwat J., Chmielewski G., Stando R., Sadowski J., Macek P., Kowalik A., Nowak-Ozimek E, & Góźdź S. (2023). Next-Generation Sequencing-Based Analysis of Clinical and Pathological Features of PIK3CA-Mutated Breast Cancer. Diagnostics, 13(18), 2887.
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10

Validating Multiplex PCR-NGS Assay for cfDNA Analysis

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Cross-platform validation was performed in 28 patients with M-Seq confirmed SNV(s)
within one or more hotspots targeted by a generic multiplex PCR-NGS panel (Extended Table 2a-b, Supplementary Table 8).
20ng of isolated cfDNA was used for library preparation using the
Oncomine™ Lung cfDNA Assay (ThermoFisher Scientific), according to the
manufacturer’s instructions. Automated template preparation and chip
loading was conducted on the Ion Chef™ instrument using the Ion
520™ & Ion 530™ Kit-Chef (ThermoFisher Scientific).
Ultimately, samples were sequenced on Ion 530™ chips using the Ion
S5™ System (ThermoFisher Scientific). Sequencing data was accessed on the
Torrent suite v5.2.2. Reads were aligned against the human genome (hg19) using
Alignment v4.0-r77189, and variants were called using the coverage Analysis
v4.0-r77897 plugin. All 18 bespoke-panel ctDNA negative patients had no tumor
SNVs detectable in plasma pre-operatively by the generic panel supporting
biological specificity of the bespoke targeted approach, 7 of 10 bespoke-panel
ctDNA positive patients had tumor SNVs detected in plasma by the generic panel
(Extended Table 2a-b). SNVs detected
by hotspot panel not identified by M-Seq are displayed in Extended Table 2c.
Abbosh C., Birkbak N.J., Wilson G.A., Jamal-Hanjani M., Constantin T., Salari R., Le Quesne J., Moore D.A., Veeriah S., Rosenthal R., Marafioti T., Kirkizlar E., Watkins T.B., McGranahan N., Ward S., Martinson L., Riley J., Fraioli F., Al Bakir M., Grönroos E., Zambrana F., Endozo R., Bi W.L., Fennessy F.M., Sponer N., Johnson D., Laycock J., Shafi S., Czyzewska-Khan J., Rowan A., Chambers T., Matthews N., Turajlic S., Hiley C., Lee S.M., Forster M.D., Ahmad T., Falzon M., Borg E., Lawrence D., Hayward M., Kolvekar S., Panagiotopoulos N., Janes S.M., Thakrar R., Ahmed A., Blackhall F., Summers Y., Hafez D., Naik A., Ganguly A., Kareht S., Shah R., Joseph L., Quinn A.M., Crosbie P., Naidu B., Middleton G., Langman G., Trotter S., Nicolson M., Remmen H., Kerr K., Chetty M., Gomersall L., Fennell D., Nakas A., Rathinam S., Anand G., Khan S., Russell P., Ezhil V., Ismail B., Irvin-sellers M., Prakash V., Lester J., Kornaszewska M., Attanoos R., Adams H., Davies H., Oukrif D., Akarca A.U., Hartley J.A., Lowe H.L., Lock S., Iles N., Bell H., Ngai Y., Elgar G., Szallasi Z., Schwarz R.F., Herrero J., Stewart A., Quezada S.A., Peggs K.S., Van Loo P., Dive C., Lin J., Rabinowitz M., Aerts H.J., Hackshaw A., Shaw J.A., Zimmermann B.G, & Swanton C. (2017). Phylogenetic ctDNA analysis depicts early stage lung cancer evolution. Nature, 545(7655), 446-451.
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