What sets these instruments apart from the others? We asked our Field Application Specialist, David O’Neill, PhD what he thought.
As a scientist, I want a system that meets the typical quality control parameters of experimentation. Reliability. Accuracy. Sensitivity. Reproducibility. I want confidence in my technology of choice. On a practical level, I want a streamlined workflow that enables me to focus my time on the analysis and interpretation of data. We all yield to strict budget lines, so I also need an affordable system that does not compromise on the factors mentioned above.
In a nutshell, the Ion S5™ and Ion S5™XL offer the simplest NGS workflow for targeted sequencing, the fastest run time, the lowest capital investment and the lowest DNA/RNA input requirements of just 1-10 ng when using Ion AmpliSeq™ technology.
Further information on the Ion Torrent™ technology can be found here.
The Ion S5™ and Ion S5™XL look identical to each other and uphold the smooth corporate image we’ve come to know over the past number of years. While they also display identical sequencer characteristics, they differ internally by their processors and graphical computing units.
The S5™ systems are not replacing their Ion siblings, the Ion PGM™ and Ion Proton™. Instead, they span the gap between the two. This is made possible thanks to the three new chips that are on offer; Ion 520™, Ion 530™ and Ion 540™ (Figure 3). The Ion 520™ chip performs similarly to the Ion PGM™ 318™ chip with 200 bp or 400 bp reads, while an Ion 530™ chip delivers three times the output of a 318™ chip, again with 200 bp or 400 bp reads. The top-end Ion 540™ chip is most similar to a Proton™ PI™ chip and supports 200 bp reads.
The different chip configurations will enable you to run both smaller panels and larger panels on the same instrument, generating outputs of 3-80 million reads with run times of just 2.5-4 hours, depending on your read length. The extra computing power of the Ion S5™XL is realized during the data analysis stage that can be completed in just 1-5 hours, which is 3-4X faster than the Ion S5™. With that, we can see how the Ion S5™XL is a great choice for labs that either need results within 24 hours and/or have a high weekly throughput. If you don’t have those pressures, the Ion S5™ is the better choice for you.
Some example research areas and applications can be seen here and popular panels for the S5™ systems can be found here.
Using a high definition and intuitive touchscreen, the user is guided through the necessary steps to prepare the instrument for sequencing and to start a sequencing run.
Unlike the Ion PGM™ and Ion Proton™, there is no need for a gas supply or specific water system to be in your lab to use an S5™. Instead, the S5™ systems use an internal pump that delivers compressed air through the system. The reagents, which are universal for any S5™ chip type and read length, are pre-made in disposable plastics and can be loaded on to the system in minutes. There’s a bottle of Wash Solution, a bottle of Cleaning Solution and a Reagents Cartridge (Figure 4). The plastics are designed in such a way as to ensure that they are inserted correctly. The systems also use RFID technology to verify that the correct reagents are loaded on the instrument.
As with the other Ion systems, the presence of atmospheric carbon dioxide can affect the pH of solutions used during sequencing. The stability of pH is a critical factor in the performance of these systems. How is this addressed with an S5™? The Reagents Cartridge not only contains the nucleotides, but also has a clever feature that will remove CO2 from the compressed air and prevent its entry to the fluidics system inside the instrument.
At this stage, the longest time is needed for allowing the reagent cartridge, stored at -20 °C, to come to room temperature before installation. The ease-of-setup is not only a time-saving benefit. It also helps greatly towards improving reproducibility between your sequencing runs and minimising potential variation as a result of user-influence – a feature we’ve seen on the Applied BioSystems 3500 Genetic Analyzers.
Finally, once the reagents are in place, the instrument performs a reagent check to ensure the system is ready to go.
At this point, you will have (i) constructed your libraries (i.e. –multiplex PCR to amplify your DNA using Ion AmpliSeq™ primer pools, ligate barcoded adaptors if pooling libraries), (ii) prepared the template (i.e. – clonal amplification of libraries on to Ion Sphere™ particles/ beads by emulsion PCR) and (iii) loaded the chip (i.e. – add the beads that contain template to the chip). This process can now be fully automated thanks to a recent configuration update on the Ion Chef™ that enables it to perform library preparation as well as template preparation and chip loading. You can load eight DNA samples on to the Ion Chef™ and in 5-7 hours the output will be one tube of normalized, pooled libraries that will have taken you about 10 pipetting steps and less than 8-10 minutes of set-up time. Of course, you can still construct your libraries by conventional means and then prepare your template using the Ion OneTouch™ 2 and load the chip manually.
Once you have your prepared chip, you are guided by instructions on the touchscreen on how to load the chip on to the instrument. The chip clamp is temperature controlled to favour the optimized sequencing chemistry and therefore reduce potential run-to-run variability.
Appearing on the instrument will be a planned run that you created in the Torrent Suite™ Software (where you entered sequencing run information, sample IDs, chip IDs and data analysis preferences). You simply select your particular protocol and then start your sequencing run.
Once your sequencing is ten completed, the next step is the data analysis. While this may seem daunting, the Torrent Suite™ Software makes it easy to get started, whether you’re a new or experienced user.
It is an easy-to-use software that is installed on the Torrent Server. You access the software through the web-based Torrent Browser. Here, you can monitor your run in real time and review run statistics and results in a standard report that generates useful graphics to show some key performance metrics, such as chip loading, number of reads and read lengths.
The Torrent Suite™ Software is optimized for the analysis of raw data using the Torrent Variant Caller (TVC) plug-in. Plug-ins enable you to expand and customize your analysis. The TVC plug-in, for example, calls single- and multi-nucleotide polymorphisms, insertions and deletions in a sample across a reference or within a subset of that reference. Additional Ion Torrent™ validated and community-developed plug-ins are available through the Ion Community.
The Torrent Suite™ Software also integrates with the Ion Reporter™ Software, the Ion Reporter™ Server, and the Torrent Suite™ Storage System (used to store sequencing runs and data results [e.g. – BAM, FASTQ]). The Ion Reporter™ Software is effectively a cloud-based toolbox that helps you streamline your data analysis. It automates the (i) importation of your sequencing data to the cloud or local Ion Reporter™ Server, (ii) analysis and identification of variants, (iii) annotation of variants from > 20 public databases or custom annotations, (iv) reporting and (v) archiving of your sequencing data. You can then export your variants in reports, spreadsheets or in text format. You can even identify complementary CE and TaqMan Assays for particular variants of interest.
Shortly after the launch of the Ion S5™ and S5™ XL, I had the opportunity to travel to Thermo Fisher Scientific’s training centre in Darmstadt, Germany. There, I got my hands on a new S5™ and combined it with an Ion Chef™ to perform the entire automated workflow. Having done similar for the Ion Proton™, where I used both the Ion OneTouch™ 2 for templating and enriching followed by manual loading, then compared this to the Ion Chef™ for templating and automatic loading, and also taking preparation of the instrument in to account, I can completely appreciate how the S5™ systems are set apart. The entire process was straightforward, uncomplicated and, dare I say it, effortless. The most taxing part was taking the consumables out of the foil wrapping. The instruments don’t necessarily perform better as sequencers compared to the Ion PGM™ and Ion Proton™ - performance is actually comparable. It’s not to suggest either that the Ion Proton™ and Ion PGM™ workflows are complex. Intimidating as they may be, they are no more complicated than following an extended protocol from a DNA extraction kit. With the S5™, what Thermo Fisher Scientific has done is delivered an incredibly simple NGS workflow from start to finish, which, to be fair, truly reflects the marketing “The S is for” expression.
Targeted sequencing has been shown to be highly effective in identifying candidate variants without the need for the additional time, cost and difficult data analysis associated with more comprehensive approaches such as whole-genome sequencing. Targeted sequencing is a major strength of Thermo Fisher, particularly in oncology, and as such the company is investing heavily to become the leader for fast, targeted applications suited to various research areas.
With that, we are seeing a refinement in Ion technology the whole way through from DNA to data. In the past few months alone we have seen a number of examples of this; the release of more gene panels, the previously mentioned updated configuration on the Ion Chef™ to allow you to construct your Ion AmpliSeq™ libraries, and of course the launch of the S5™ and S5™XL. There is a drive to expand the number of off-the-shelf assay panels and to optimize current panels to suit automated library preparation on the Ion Chef™. When this dynamic Ion AmpliSeq™, Ion Chef™ and Ion S5™ trio are combined, the result is the simplest and fastest targeted sequencing system that takes less than 45 minutes of hands-on time to set up.
In the first four years since its introduction, Ion Torrent™ technology has been referenced in over 1,500 publications, which is essentially a rate of one publication per day. With these recent developments and Thermo Fisher’s evident commitment to bring innovation and continual improvements to aid scientists in their research, there’s no doubt that we will see the application of Ion Torrent™ expand within and across research areas.
David O’Neill, PhD
Field Application Specialist