Ion sphere quality control kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion Sphere Quality Control Kit is a laboratory tool designed to verify the quality and performance of Ion Sphere Particles used in Ion Torrent sequencing systems. The kit provides a standardized sample to assess key parameters critical for successful sequencing runs.

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Sphere quality control kit (4 citations) Ion Spheres (2 citations) Ion Sphere™ Quality Control Kit assessment (2 citations)

31 protocols using ion sphere quality control kit

Ion Torrent Sequencing Library Preparation

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Sequencing libraries were quantified with quantitative polymerase chain reaction (qPCR) with the Ion Library Quantitation Kit (Cat # 4,468,802, Life Technologies) and diluted to 100 pM. The diluted sequencing libraries (10 pM) were then amplified on Ion Sphere^TM Particles (ISPs) and enriched for template-positive ISPs using the Ion PGM Template OT2 200 Kit (Cat # 4,480,974), Ion One Touch 2 instrument, and Ion One Touch ES. The Ion Sphere Quality Control Kit (Cat # 4,468,656, Life Technologies) was used to determine the fraction of template-positive ISPs. Enriched ISPs were then sequenced using the Ion PGM Hi-Q sequencing Kit (Cat # A25592, Life Technologies) and the 318 Chip Kit v2 (Cat # 4,484,354, Life Technologies). Ion Torrent sequencing results were deposited in the NCBI Sequence Read Archive with study accession number SRP067259.

Collins D.W., Gudiseva H.V., Trachtman B., Bowman A.S., Sagaser A., Sankar P., Miller-Ellis E., Lehman A., Addis V, & O'Brien J.M. (2016). Association of primary open-angle glaucoma with mitochondrial variants and haplogroups common in African Americans. Molecular Vision, 22, 454-471.

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Microbiome Profiling of Soil and Plant Compartments

Cited 2 times

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The V4-V5 regions of the 16S rRNA gene and the ribosomal internal transcribed spacer 2 (ITS2) from the rhizosphere, bulk soil, root, and seedbed were PCR-amplified to characterize the bacterial and fungal microbiota, respectively. The 16S RNA gene was amplified with the primer pair 520 F (5′-AYTGGGYDTAAAGNG-3′) (Claesson et al., 2009 (link)) and 907 R (5′-CCGTCAATTCMTTTRAGTTT-3′) (Engelbrektson et al., 2010 (link)) in combination with peptide nucleic acid (PNA) clumps, and purified, as described by Abdullaeva et al. (2021) (link). The primer pair for the fungal ITS2 regions was ITS3 KYO2 forward (5′-GATGAAGAACGYAGYRAA-3′) and ITS4 reverse (5′-TCCTCCGCTTATTGATATGC-3′); amplification and purification were performed as described by Ambika Manirajan et al. (2018) (link).
Polymerase chain reaction products were pooled in equimolar concentrations and used for emulsion PCR with Ion One Touch 2 (Ion PGM Hi-Q View OT2 kit, Life Technologies, Carlsbad, CA, United States). The quality of the final product was assessed using Ion Sphere Quality Control Kit (Life Technologies, Carlsbad, CA, United States) and loaded on a 314 or 318 chip for sequencing with an Ion PGM sequencer (Life Technologies, Carlsbad, CA, United States).

Abdullaeva Y., Ratering S., Ambika Manirajan B., Rosado-Porto D., Schnell S, & Cardinale M. (2022). Domestication Impacts the Wheat-Associated Microbiota and the Rhizosphere Colonization by Seed- and Soil-Originated Microbiomes, Across Different Fields. Frontiers in Plant Science, 12, 806915.

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Ion Torrent Sequencing Workflow

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The template preparation was performed according to the Ion OneTouch System protocol (PNMAN0006957, Rev. 6.0) according to manufactory instructions. Briefly, an input concentration of one DNA template copy per Ion Sphere Particle (ISP) was added to the emulsion PCR master mix. The emulsion preparation was followed by DNA clonal amplification using the Ion OneTouch instrument (Life Technologies). The ISPs were then recovered, and the template-positive ISPs were enriched using Dynabeads MyOne Streptavidin C1 beads (Life Technologies) in the Ion OneTouch ES instrument (Life Technologies). ISP enrichment was qualified using the Ion Sphere quality control kit (Life Technologies) and a Qubit 2.0 fluorometer (Life Technologies). The complete ISP samples were loaded on either Chip 314 or Chip 316 and prepared for sequencing using the Ion PGM 200 sequencing kit protocol (PN4474246, Rev. D). The sequencing runs were performed using 520 flows for 130 cycles.

Gu Y., Lu K., Yang G., Cen Z., Yu L., Lin L., Hao J., Yang Z., Peng J., Cui S, & Huang J. (2014). Mutation Spectrum of Six Genes in Chinese Phenylketonuria Patients Obtained through Next-Generation Sequencing. PLoS ONE, 9(4), e94100.

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Ion Torrent PROTON System Sequencing

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Clonal amplification of the bar-coded DNA library onto ion spheres (ISPs) was conducted using emulsion PCR, and the DNA of the ISPs was subsequently isolated using an Ion PI HIQ OT2 200 kit and Ion OneTouch ES (Life Technologies) according to the manufacturer's instructions. We determined the polyclonal percentages and quality of enriched, template-positive ISPs using an Ion Sphere Quality Control kit (Life Technologies). We then sequenced samples with polyclonal percentages <30% and enriched, template-positive ISPs >80% on the Ion Torrent PROTON system (Life Technologies).

Park J., Shin S.Y., Kim K., Park K., Shin S, & Ihm C. (2018). Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates. Annals of Laboratory Medicine, 38(4), 316-323.

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Ion Xpress DNA Library Preparation Protocol

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The genome library preparation was performed by using the Ion Xpress^TM Plus Fragment Library Kit (Life Technologies, USA) following the manufacturer’s protocol, 4471989 Rev E. The sheared DNA was separated using a 2% agarose gel (Promega, USA) and fragments that corresponded to 200 bp of a sequencing run (approximately 330 bp) were manually excised. The DNA was purified using the QIAquick Gel Extraction Kit (Qiagen, Germany). The library quality and concentration were assessed and determined using the 2100 Bioanalyzer (Agilent, USA) and High Sensitivity DNA Kit (Agilent, USA). The sequencing template was prepared using the Ion OneTouch^TM 200 Template Kit V2 DL (Life Technologies, USA) according to the manufacturer’s protocol. The Ion Sphere Particles were enriched using Dynabeads MyOne Streptavidin C1 beads (Invitrogen, Life Technologies, USA). The efficiency of the enrichment process was assessed using the Ion Sphere Quality Control Kit (Life Technologies, USA). Genome sequencing was undertaken using the Ion Torrent PGM sequencer (Life Technologies, USA).

Tan K.K., Tan Y.C., Chang L.Y., Lee K.W., Nore S.S., Yee W.Y., Mat Isa M.N., Jafar F.L., Hoh C.C, & AbuBakar S. (2015). Full genome SNP-based phylogenetic analysis reveals the origin and global spread of Brucella melitensis. BMC Genomics, 16(1), 93.

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MeDIP Library Preparation and Ion Sequencing

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MeDIP libraries were templated using the Ion OneTouch 2 System instrument with either the Ion PGM Template OT2 200 kit for the PGM instrument or the Ion PI Template OT2 Kit v2 for the Proton instrument according to manufacturer’s protocols (Life Technologies). Following template reactions, templated libraries were assessed for quality using the Ion Sphere Quality Control Kit (Catalog No. 4468656) and Qubit fluorometer (Life Technologies). Templated libraries that passed manufacturer recommended criteria were enriched using the Ion OneTouch ES instrument. Immediately after enrichment, MeDIP libraries were sequenced on either an Ion PGM or Ion Proton Semiconductor Sequencing machine (Life Technologies).

Corley M.J., Vargas-Maya N., Pang A.P., Lum-Jones A., Li D., Khadka V., Sultana R., Blanchard D.C, & Maunakea A.K. (2019). Epigenetic Delay in the Neurodevelopmental Trajectory of DNA Methylation States in Autism Spectrum Disorders. Frontiers in Genetics, 10, 907.

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Ion Torrent PGM Library Preparation

Cited 1 time

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We used the methodology previously described by Pisarenko et al. [33 (link)]. The preparation of a genomic library with a 400-bp read length was performed using the Ion Xpress Plus Fragment Library Kit reagent kit (Life Technologies, USA) in accordance with the manufacturer’s protocol. DNA library fragments were separated using a ready-to-use commercial kit 2% E-Gel SizeSelect agarose gel (Invitrogen, USA). The finished library of DNA fragments was purified using Agencourt AMPure XP magnetic particles (Beckman Coulter, USA). Library quality and concentration were determined using the Experion™ Automated Electrophoresis System and Experion DNA 1 K Reagents and Supplies and Experion DNA Chips kits (Bio-Rad, USA). Monoclonal amplification on microspheres was performed using Ion PGM Hi-Q View OT2 Kit reagents (Life Technologies, USA) in accordance with the manufacturer’s protocol. Microsphere enrichment was performed using Dynabeads MyOne Streptavidin C1 magnetic particles (Invitrogen, Life Technologies, USA). The effectiveness of the enrichment process was evaluated using the Ion Sphere Quality Control Kit (Life Technologies, USA). Genome sequencing was performed using an Ion Torrent PGM sequencer and Ion 316 Chips Kit V2 chips (Life Technologies, USA).

Pisarenko S.V., Eremenko E.I., Ryazanova A.G., Kovalev D.A., Buravtseva N.P., Aksenova L.Y., Evchenko A.Y., Semenova O.V., Bobrisheva O.V., Kuznetsova I.V., Golovinskaya T.M., Tchmerenko D.K., Kulichenko A.N, & Morozov V.Y. (2019). Genotyping and phylogenetic location of one clinical isolate of Bacillus anthracis isolated from a human in Russia. BMC Microbiology, 19, 165.

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Ion Proton MeDIP Library Preparation

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MeDIP libraries were templated using the Ion OneTouch 2 System instrument with the Ion PI Template OT2 Kit v2 for the Ion Proton instrument according to manufacturer's protocols (Life Technologies). Following template reactions, templated libraries were assessed for quality using the Ion Sphere Quality Control Kit (Catalog # 4,468,656) and Qubit fluorometer (Life Technologies). Templated libraries that passed manufacturer recommended criteria were enriched using the Ion OneTouch ES instrument. Immediately after enrichment, MeDIP libraries were sequenced on the Ion Proton with PI chips using the Ion PGM 200 Sequencing kit or Ion PtdIns Sequencing 200 Kit v2, according to instructions provided by the manufacturer (Life Technologies).

Lees J., Pèrtille F., Løtvedt P., Jensen P, & Bosagna C.G. (2023). The mitoepigenome responds to stress, suggesting novel mito-nuclear interactions in vertebrates. BMC Genomics, 24, 561.

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Ion PGM Amplicon Sequencing Protocol

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After making a pooled, equimolar library, 400 bp (amplification) emulsion PCR
(emPCR) was conducted using the Ion Personal Genome Machine (PGM) Hi-Q View OT2
Kit (Life Technologies Corp.; Cat. no. A29900). Quality of the emPCR was checked
using the Ion Sphere Quality Control Kit (Life Technologies Corp.; Cat. no.
446856). The 400 bp library was assessed using the Ion Sphere Assay with a Qubit
3.0 fluorometer (Life Technologies Corp.; MAN0016388) and passed with 32.50%
templated Ion Sphere Particles (ISPs). Enrichment of template-positive Ion PGM
Hi-Q View ISPs was done using the Ion OneTouch ES following manufacturer
protocol (Life Technologies; MAN0014579). The enriched library was then
processed for DNA sequencing. The 400 bp sequencing was done on the Ion PGM
using the Hi-Q View Sequencing Kit (Life Technologies Corp.; Cat. no. A30044)
using an Ion Torrent 318 v2 BC chip and allowing for 850 flows of sequencing
dNTPs (Life Technologies Corp.; Cat. no. 4488146).

McDermid K.J., Kittle RP I.I.I., Veillet A., Plouviez S., Muehlstein L, & Balazs G.H. (2020). Identification of Gastrointestinal Microbiota in Hawaiian Green Turtles (Chelonia mydas). Evolutionary Bioinformatics Online, 16, 1176934320914603.

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Ion Sphere Particle Generation

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A diluted library (26 pM) was used to generate template positive Ion Sphere™ Particles (ISPs) containing clonally amplified DNA. Emulsion PCR was conducted using the OneTouch™ 200 Template Kit v2 DL with the Ion OneTouch™ DL configuration (Life Technologies), template-positive ISPs were enriched with the Ion OneTouch™ ES (Life Technologies), and quality of template-positive ISPs was assessed by using the Ion Sphere™ Quality Control Kit (Life Technologies) on the Qubit® 2.0 Fluorometer, following the recommended protocol.

Seo S.B., Zeng X., King J.L., Larue B.L., Assidi M., Al-Qahtani M.H., Sajantila A, & Budowle B. (2015). Underlying Data for Sequencing the Mitochondrial Genome with the Massively Parallel Sequencing Platform Ion Torrent™ PGM™. BMC Genomics, 16(Suppl 1), S4.

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