Quality Control of the Ion Sphere™ Particles (ISPs)

A new assay has recently been developed for the Qubit® 2.0 Fluorometer that enables quick and easy assessment of template-positive ISPs prior to performing a sequencing run. All ISPs bind a complementary, green-fluorescent Alexa Fluor® 488 dye–labeled oligonucleotide, whereas only those beads that are template-positive bind a complementary red-fluorescent Alexa Fluor® 647 dye–labeled oligonucleotide (Figure 1). The Qubit® 2.0 Fluorometer measures the fluorescence emission of each dye conjugate, and a Microsoft Excel® file template is used to calculate the percentage of template-positive ISPs from the ratio of the Alexa Fluor® 647 dye signal to the Alexa Fluor® 488 dye signal.

This quality-control step using the Qubit® 2.0 Fluorometer determines the likelihood of a successful sequencing run, ultimately saving time and expense during subsequent sequencing on the Ion PGM™ Sequencer. The assay is described in the template preparation user guides for the Ion semiconductor sequencing workflow, which can be found at the Ion Community website (ioncommunity.iontorrent.com). Necessary firmware and plug-in files for this robust and reproducible assay can be downloaded from the Ion Community using the USB drive provided with the Qubit® 2.0 Fluorometer. This new application extends the functionality of the Qubit® 2.0 Fluorometer from DNA, RNA, and protein quantitation to quality control for semiconductor sequencing.

Figure 1. The Ion Sphere™ Particle (ISP) assay principle using Alexa Fluor® dyes. The B nucleic acid sequence is present in both the ISP primer and the template and is used to drive the amplification of the template onto the bead; the A sequence is only present in the template. The ratio of fluorescence signals from Alexa Fluor® 647 and Alexa Fluor® 488 dye–labeled oligonucleotides is used to determine the relative amount of template-positive particles.

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The Qubit® 2.0 Fluorometer provides a simple, fast, and robust workflow for accurate sample measurement using predefined assay parameters in conjunction with “mix and read” Qubit® assays. The flexibility of the Qubit® 2.0 Fluorometer and associated assays expands the breadth of applications that can be done on this low-cost platform, providing a great resource for your application needs and enabling faster time-to-results. Find out more about the Qubit® 2.0 Fluorometer, the Ion PGM™ Sequencer, and our other benchtop instruments.