NGS Template Preparation Support—Getting Started

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.  

Here are the kit components:

Ion PGM Template IA Supplies 500 (Part No. A24618)
- Ion OneTouch ES Supplies: 1 bag

Ion PGM Template IA Reagents 500 (Part No. A24619)
- Ion PGM Template IA Pellets 500: 4 foil pouches [1]
- Ion PGM Template IA ISP Dilution Buffer (yellow cap): 1 mL
- Ion PGM Template IA Start Solution (purple cap): 2 x 1200 µL

Ion PGM Template IA Reactions 500 (Part No. A24620)
- Ion PGM Template IA Ion Sphere Particles (orange cap): 86 µL
- Ion PGM Template IA Primer Mix S (black cap): 48 µL
- Ion PGM Template IA Primer Mix L (blue cap): 48 µL [2]
- Ion PGM Template IA Rehydration Buffer (white cap): 2 x 1.5 mL

Ion PGM Template IA Solutions (Part No. A24621)
- Ion PGM Template IA Stop Solution: 2.8 mL
- Ion PGM Template IA Recovery Solution: 7 mL
- Ion PGM Template IA Wash Solution: 4 mL
- MyOne Beads Wash Solution (green cap): 1.04 mL
- Neutralization Solution (red cap): 40 µL
- Tween Solution: 3.5 mL

Note: [1] One Ion PGM Template IA Pellet 500 per tube per pouch.
[2] Primer Mix L is intended for amplification of long templates and is not used with the Ion ReproSeq PGS View Kit

Visible Ion Sphere particle (ISP) beads help minimize user variability in manual chip loading, to help improve experimental reproducibility and confidence in your results. Additionally, the new Ion Sphere particles have been optimized to provide increased sequencing performance.

Yes, the Ion PGM Hi-Q View sequencing chemistry is compatible with Ion ReproSeq PGS kits.

Yes, the Ion PGM Hi-Q View chemistry is compatible with both 200 bp and 400 bp libraries.

You will need Torrent Suite Software v5.0.4 or later on the Ion PGM System.

In addition to updating the Ion OneTouch 2 System firmware to Torrent Suite Software v.5.0.4 or later, you will have to perform the chemistry change protocol. To set up the Ion OneTouch 2 Instrument when switching between chemistries, refer to Chapter 5 of the Ion PGM OneTouch 2 Hi-Q View user guide,“Demonstrated Protocol: Setting up the Ion OneTouch 2 Instrument when switching between template preparation kits”.

No, the Ion PGM Hi-Q View OneTouch 2 Kit has only been validated with the Ion PGM Hi-Q View Sequencing Kit.

From sample to sequence, it takes less than 12 hours.

The IA step takes less than two hours, with less than 30 minutes of hands on time.

The Ion PGM Template IA 500 Kit is compatible with up to 500 bp read libraries, with a total library length of 580 bp (including adapters).

The Ion PGM Template IA 500 Kit is compatible with the Ion PGM Hi-Q View Sequencing Kit. It is not compatible with the Ion PGM Hi-Q Sequencing Kit.

Torrent Suite Software v.5.0.4 or higher is needed.

No, at this time it is only compatible with the Ion One Touch ES instrument.

No, the Ion PGM Template IA 500 Kit is only compatible with libraries that have a full length P1 adapter.

The Ion PGM Template IA 500 Kit is not compatible with the Ion Sphere Quality Control Kit.

You may expect 60-80 million reads with an Ion 540 chip.

The Ion 540 chip is compatible with the Ion GeneStudio S5/S5 Plus/Prime and Ion S5/S5 XL instruments.

You may expect 100-130 million reads with an Ion 550 chip.

Remove and sequence chips as soon as possible after the Ion Chef System finishes loading them. If you cannot sequence a loaded chip immediately, store it in a chip storage container at 4 degrees C until you are ready to run it (up to 24 hours maximum).

The Ion 550 Single Chip Supplemental Kit contains pre-packaged templating supplies and sequencing reagent cartridges required to support eight single chip Ion Chef runs and eight sequencer initializations with the Ion 550 Kit-Chef. The Ion 550 Single Chip Supplemental Kit includes the following components:

- Ion S5 Chef Supplies (4 boxes) (ships at room temperature; store at room temperature)
- Ion S5 Sequencing Solutions (1 box) (ships at room temperature; store at room temperature)
- Ion S5 Sequencing Reagents (1box) (ships on wet ice at 2-8 degrees C; store at -20 degrees C)
IMPORTANT! Do not store the Ion S5 Sequencing Reagents (Part No. A27768) on dry ice or in a closed environment containing dry ice.

The Ion 550 Kit-Chef contains Ion Chef templating and sequencing consumables sufficient for preparing and sequencing eight Ion 550 chips for four Ion Chef runs and four sequencer initializations (dual chip run configuration). If you are running in the single chip configuration, then you will need to purchase the Ion 550 Single Chip Supplemental Kit separately to ensure that you have enough templating and sequencing consumables to support eight single chip Ion Chef templating runs and eight sequencer initializations with the Ion 550 Kit-Chef.

When preparing the template preparation reaction, add 10 µL of the Ion PGM Calibration Standard to the amplification solution, and reduce the volume of Nuclease-free water added to 15 µL. When creating your Planned Run in the Torrent Browser, select "Enable Calibration Standard" from the Base Calibration Mode dropdown list in the Kits tab.

The kit provides components for 8 initializations (8 runs at 1 run per initialization) on the Ion S5 system.

The kit provides components for 8 initializations (8 runs at 2 runs per initialization) on the Ion S5 system.

The kits are compatible with the following commercially available Precision ID panels: GlobalFiler NGS STR Panel v2, Identity Panel, Ancestry Panel, mtDNA Whole Genome Panel, and mtDNA Control Region Panel.

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 run should be completed in approximately 1 hour. 

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

The components of the Ion 520 & 530 Kit - Chef can be found here.

The shelf life of the Ion 520 & 530 Kit - Chef is 12 months from the date of manufacture.

The Ion 520 & 530 Kit - Chef (Cat. No. A27757) contains reagents and consumables for performing 8 runs, either 200 bp or 400 bp (1 run per initialization). The Ion 520 & 530 Kit - Chef (Cat. No. A30010) contains reagents and consumables for performing 8, 200 bp runs (2 runs per initialization).

No, you must run two of the same types of chips in the same Ion Chef run.

Two Ion 520 or 530 chips can be prepped and loaded in 13 hours, with only 15 minutes of hands-on time.

It is possible to prepare just one chip on the Ion Chef Instrument per run, utilizing the Ion S5 Chip Balance, which is included in the Ion S5 Installation Kit. Please note that the Ion Chef Instrument will still use up reagents for two chips.

We recommend sequencing the second chip within 8 hours after the Ion Chef Instrument run has finished.

The components of the Ion 540 Kit - Chef can be found here.

The shelf life of the Ion 540 Kit - Chef is 12 months from the date of manufacture.

Two Ion 540 chips can be prepped and loaded in 13 hours, with only 15 minutes of hands on time.

No, the Ion 16S Metagenomics Kit utilizes 400 bp reads, so it is incompatible with the Ion 540 Kit - Chef, which only yields 200 bp reads.

The Ion 550 Kit-Chef includes reagents and materials that are required to use the Ion Chef System to prepare template-positive Ion Sphere particles from up to 200-base-read libraries and load eight Ion 550 chips (two chips/run). The kit also includes reagents and materials to sequence eight loaded chips (two sequencing runs/initialization).

The Ion 550 Kit-Chef is supported on the Ion GeneStudio S5 Plus System and Ion GeneStudio S5 Prime System. The Ion 550 Kit-Chef is also compatible with the Ion S5 XL System, but is not compatible with the Ion S5 System, nor the base model Ion GeneStudio S5 System.

The Ion 550 Kit-Chef can be used with up to 200-base-read libraries of any type, prepared using any available Ion library kit.