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Template Preparation

Ion Torrent™ OneTouch™ 2 protocols can be found in our NGS Template Preparation Support Center. For all other protocols, please go here.

The calibration standard is an optional reagent and is recommended for samples that have no reference or small references. It is a small panel consisting of known sequence containing homopolymers. It provides a reference for base calibration but the option needs to be selected in run planning.

The Ion Sphere Quality Control assay on the Qubit 2.0 Fluorometer labels the Ion Sphere Particles (ISPs) with two different fluorophores: Alexa Fluor™ 488 and Alexa Fluor 647. The probe labeled with Alexa Fluor 488 anneals to primer B sites, or all of the ISPs present. The probe labeled with Alexa Fluor 647 anneals to primer A sites, or only the ISPs with extended templates.

The ratio of Alexa Fluor 647 fluorescence (templated ISPs) to Alexa Fluor 488 fluorescence (all ISPs present) gives the percent templated ISPs.

Please see Appendix A of your template user guide for further information.

The Qubit conversion factor can be found here. Please use the lot number from the Reagents Kit box to look up the corresponding conversion factor.

Firmware updates for the Ion OneTouch 2 instrument are included in the Torrent Suite Software updates (the Ion OneTouch ES instrument does not require firmware updates). After updating the Torrent Server, the Ion OneTouch 2 firmware can be updated by downloading the firmware from the Torrent Server to a USB drive or through a direct ethernet connection to the Torrent Server. Please see the appropriate Appendix of your Ion Template kit user guide for detailed, step-by-step instructions.

The Ion ReproSeq™ PGS Kit workflow does not use the Ion OneTouch™ 2 System for template preparation.  Template preparation is performed using Ion IA technology, whereby DNA is clonally amplified onto a bead surface through a non-emulsion, isothermal reaction.

The template ISPs should be in the Ion PGM™ Template IA Wash Solution and can be stored at 4 degrees C for up to one week.

The enriched ISPs can be stored at 2 degrees C to 8 degrees C for up to 3 days.

The Ion PGM™ Hi-Q™ OneTouch™ 2 Kit includes the reagents required for preparing 8 reactions of template-positive Ion PGM™ Hi-Q™ Ion Sphere™ Particles (ISPs) on the Ion OneTouch™ 2 System.  The components are:

  • Ion PGM™ OneTouch™ 2 Supplies
  • Ion PGM™ Hi‑Q ™ OneTouch™ 2 Reagents
  • Ion PGM™ Hi‑Q ™ OneTouch™ 2 Solutions
  • Ion PGM™ Calibration Standard

The calibration standard is an optional reagent and is recommended for samples that have no reference or small references.  It is a small panel consisting of known sequence containing homopolymers.   It provides a reference for base calibration but the option needs to be selected in run planning.  

Ion Chef™ System

The Ion Chef System simplifies the workflow for Ion PGM and Ion Proton systems, providing an automated, high-throughput research solution for reproducible template preparation and chip loading. For more information, click here.

No, the Ion Chef utilizes all of the reagents in each run regardless of the number of chips being prepared, therefore we recommend always preparing two chips to maximize the throughput of the Ion Chef.

The Ion Chef instrument provides two options for obtaining quality control (QC) samples that can be used to assess templating efficiency. Depending on your selection, the QC samples will be made available either during or after the run. In either case, you can obtain unenriched samples from the corresponding Library Sample Tubes at Positions A and B on the Ion PGM Hi-Q Chef Reagents Cartridge, or enriched samples from Positions A and E on the Enrichment Cartridge v2.

The run time is approximately 10.5 hours for 200-base read sequencing and about 14 hours for 400-base read sequencing. 

The components are listed below:

  • Ion PI™ Hi-Q™ Chef Supplies – 4 boxes
  • Ion PI™ Hi-Q™ Chef Solutions – 4 cartridges
  • Ion PI™ Hi-Q™ Chef Reagents – 4 cartridges
  • Ion Proton™ Sequencing Supplies – 1 box
  • Ion PI™ Hi-Q™ Sequencing 200 Solutions – 1 box
  • Ion PI™ Hi-Q™ Sequencing  Nucleotides – 1 box

The main improvements are:

  • Higher sensitivity due to improved templating reaction conditions and improved amplification efficiency, especially of GC-rich regions
  • Higher specificity and higher amplicon end-to-end sequencing due to the novel Ion Hi-Q™ sequencing enzyme
  • Reduction in hands-on time by 45 minutes due to the pretreatment of the Ion PI Chips v3 in manufacturing
  • Over 60% reduction in input DNA required for templating
  • Two sequencing runs per initialization