Enabling New Workflows With the Qubit® 2.0 Fluorometer
A Quality Control Assay for Ion Torrent™ Semiconductor Sequencing
Powered by Ion Torrent™ semiconductor chip technology, the Ion Personal Genome Machine® (PGM™) Sequencer is a next-generation benchtop instrument that offers fast, affordable sequencing. This system sequences DNA templates that have been clonally amplified on primer-coated beads, known as Ion Sphere™ Particles (ISPs). Traditionally, confirmation of template-positive ISPs prior to a sequencing run has been performed using microscopy or flow cytometry. However, systematic quality control of the templated beads has largely been ignored due to high costs in terms of both time and money—until now.
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.
Learn More About Our Benchtop Instruments
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.
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