Ion Sphere™ Quality Control Kit - FAQs

For sequencing on the Ion PGM instrument, we recommend that the percent templated ISPs for your unenriched ISP sample be ~10-30% to maximize sequencing run throughput.

For sequencing on the Ion Proton instrument, we recommend that the percent templated ISPs for your unenriched ISP sample be ~10-25% to maximize sequencing run throughput.

Samples that fall within the recommended range generally produce the most data; however, samples that fall outside of the recommended range can still meet the throughput specifications on the Ion chips. The recommended optimal range is not intended to be a pass/fail criteria, but instead, provides guidance for the quality of the sample.
• Uneneriched ISPs with < 10% templated ISPs will likely have an insufficient number of templated ISPs to achieve optimal loading density on the Ion chip. The library input into template preparation should be increased to increase the percent templated ISPs.
• Unenriched ISPs with >25% (Ion Proton) or >30% (Ion PGM) templated ISPs will likely have a greater number of polyclonal (or more than one library fragment population) ISPs. The library input into template preparation should be decreased to decrease the percent templated ISPs.

The conversion factor for each individual lot of the Ion OneTouch 2 PGM and Proton template kits can be found here (https://tools.thermofisher.com/content/sfs/brochures/qubit-conversion-factors-guide.pdf). Please use the lot number from the Reagents Kit box to look up the corresponding conversion factor.

The Ion Sphere Quality Control assay on the Qubit 2.0 or 3.0 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 the Alexa Fluor 647 fluorescence (templated ISPs) to the Alexa Fluor 488 fluorescence (all ISPs present) yields the percent templated ISPs.

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

Yes, there are three supported options:

• Ion Sphere Quality Control Kit (Cat. No. 4468656) for use with the Qubit 2.0 or 3.0 Fluorometer - This is the most commonly used templated ISP quality control method. This kit supports the quality control of unenriched ISPs and the protocol can be found in the Appendix of all Ion Template kit user guides.
• ISP quality control using flow cytometry:
- Ion Sphere Particles Quality Assessment Using the Guava easyCyte 5 Flow Cytometer (http://ioncommunity.thermofisher.com/docs/DOC-2494)br/> - Demonstrated Protocol: Ion Sphere Particles (ISPs) Quality Assessment using the Applied Biosystems Attune Acoustic Focusing Cytometer (http://ioncommunity.thermofisher.com/docs/DOC-2790)

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Yes, the Ion Sphere Quality Control Kit (Cat. No. 4468656) is compatible with Qubit fluorometers.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

The conversion factors for the ISP fluorescence readings on the Qubit 2.0 or 3.0 Fluorometer are specific to the template kit (Ion Chef kits or Ion One Touch 2 kits) and can be found online (https://assets.thermofisher.com/TFS-Assets/LSG/brochures/qubit-conversion-factors-guide.pdf). Please use the lot number from the Reagents kit box to look up the corresponding conversion factor.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

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 the Appendix of your template user guide for further information.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.