Please can you introduce yourself and tell us about your role at SPT Labtech?
My name is Huw Rees. As a member of SPT Labtech’s North American Field Application Science team, I provide expert training and application advice to users of our liquid handling solutions, including mosquito, firefly®, and dragonfly®.
What is library preparation in NGS and why automate it?
Next-generation sequencing (NGS) library preparation normally has several key parts, but the key steps that require liquid handling are shown below:
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Almost all kits require DNA end preparation, where you modify the ends to make them suitable for sequencing. This is followed by a clean-up step to remove enzymes and other reagents from the initial preparation step.
After PCR, the next step is another clean-up before placing your samples into the sequencer. These kits are ideal for automation because they are extremely laborious and time-consuming to do by hand. Preparing these kits manually, especially for plates, can take hours of precise, focused work and be difficult to master.
Experienced lab bench workers who can perform these kits rapidly, consistently, and effectively are hard to find. Automating the process reduces the need for specialized skills and allows scientists to focus on other parts of their research.
Automated liquid handlers can pipette volumes below what a handheld pipette can manage. Sub-microliter volumes are hard to do with handheld pipettes, but automation can achieve this consistently.
What are the benefits of miniaturizing NGS library preparation? How does this affect the cost per sample?
Miniaturizing NGS library preparation is important because it makes your money go further. While effective, library prep kits generally have a high cost per sample due to the expense involved in their production, such as protein engineering and extensive research.
By miniaturizing these kits, you can significantly reduce the volumes required per reaction, allowing you to process more samples with the same amount of reagent, saving money. Automation further enhances this by increasing laboratory throughput. Miniaturization also reduces your reliance on manual pipetting, provides walkaway time, and increases the throughput of samples in your workflow.
How can labs achieve more substantial miniaturization?
Substantial miniaturization can be achieved in high read-depth kits. For example, the NEBNext Ultra II DNA kits can be miniaturized about tenfold on mosquito, making your kit go ten times further to give you an idea of cost savings. Other kits can also be miniaturized to a similar extent, or even further.
Can you talk more about the mosquito and how it maximizes ROI through miniaturization?
mosquito is our nanoliter liquid handling instrument designed to maximize ROI through miniaturization. mosquito’s tips are loaded onto a spool at the top of the instrument that feeds them through into a pipetting head. This pipetting head can aspirate across five available plate deck positions, with the ability to work in both 96 and 384-well formats. The tips are made of stainless steel with a high-density polyethylene sheath, ensuring durability and precision. Each individual tip utilizes true positive displacement technology, meaning there is no air interface between the liquid and the tip. This means that accuracy isn’t compromised when working with low or high-viscosity liquids, meaning it can handle very viscous master mixes or volatile mixtures like ethanol without issues.
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mosquito can handle volumes of 500 nL down to 5 µL, with a low-volume version capable of handling 1.2 µL down to 25 nL. For this discussion, I will predominantly focus on the high-volume version as it has the capability of miniaturizing magnetic bead clean-ups.
The one-way direction of the tips virtually eliminates the chance of cross-contamination. mosquito’s ability to contact dispense and aspirate from the very bottom of the well ensures negligible dead volumes, making it very gentle on genomic DNA and enzymes. This is very important for NGS library preparations, as it avoids cavitation and vortexing, which can break havoc on enzymes.
How can mosquito be used to easily set up NGS library prep?
mosquito comes with a comprehensive software package that includes default wizards for simplifying the setup of common NGS protocols, such as Nextera XT, NEB kits, and bead clean-ups. The software also allows for custom experiment setups with the help of our FAS team, enabling labs to miniaturize kits specific to their needs. The user-friendly software interface allows easy customization and modification of experiments.
One example of a pre-loaded protocol is the NEBNext Ultra II FS DNA Kit. You will start on the Run page, which has a pre-written detailing the steps it will perform, and an estimated run time.
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If customization is needed, you can modify the experiment from the Information page, which provides the protocol’s title and description. This protocol is optimized for saving reagent tips and time. It also includes steps for fragmentation, end repair, dA tailing, adapter ligation, and USER enzyme treatment.
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The Plates tab allows you to specify the type of plates you will be using. You can swap out these defaults if needed.
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Under the Experiment tab, you can reduce the number of columns on the plate if you are running fewer samples. You also have the ability to modify where reagent columns are placed. For instance, you can move the ligation mix to a different column to reduce dead volume.
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After physically loading plates and samples onto the instrument, you simply need to hit run. mosquito will then distribute the fragmentation mix over the reaction plate, completing your NGS library prep setup.
Can you tell us more about the clean-up protocol after library preparation?
After performing the initial library preparation and preparing the DNA ends, the next step is to clean up the sample, which involves using the bead clean-up protocol. The mosquito software wizard facilitates this process from start to finish, providing a run page with all the necessary details.
This protocol involves five plates: a sample plate, a reagent plate, a position for the sample plate on the magnetic block, a waste plate, and an output place for the purified sample after elution.
Using a similar wizard, users can customize the types of plates under the Plates tab, and adjust the bead cleanup protocol in the Experiment tab, such as bead ratio, number of ethanol washes, and whether to include ethanol incubation. Users can also select the number of samples to run, and designate columns for beads, ethanol, and other reagents.
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Once the plates are set up, mosquito adds beads to the samples, and the plate is vortexed and pulse centrifuged. After incubating the beads with the sample, the plate is placed on the magnet for the ethanol washing steps, which mosquito handles by adding and removing ethanol.
The plate is then moved off the magnet, the elution buffer is added to resuspend the beads, and the plate is placed back on the magnet to ensure no beads end up in the final sample.
This protocol is just one of many workflows supported by mosquito. We have extensive citations and resources supporting full NGS workflows from beginning to end with varying levels of miniaturization.
Can you provide an example of how mosquito can reduce the cost per sample?
Two robust and well-known kits are Illumina Nextera XT and the Illumina DNA prep kits. With mosquito, we have consistently achieved tenfold miniaturization—or 12.5-fold in the case of Nextera XT—resulting in a significant reduction in cost per sample.
For instance, using 384-well sample plates only takes processing eight full plates to cover the cost of a mosquito instrument in your lab. This means that you will see a return on your investment very quickly. In some labs, this level of throughput is common, allowing mosquito to pay for itself within a matter of weeks.
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Are there any exciting mosquito updates you’d like to share?
We have just very recently launched the latest member of our mosquito family – mosquito Gen3. It’s our most compact and accessible version yet, offering all the capabilities of mosquito but with three deck positions and a smaller footprint. With Gen3, we’re lowering the barrier to miniaturization, so even more genomics researchers can benefit from the cost savings of reducing NGS volumes.
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About Huw Rees
As a member of the North American FAS team, Huw is responsible for providing expert training and application advice to users of SPT Labtech liquid handling solutions across
the Northeast US and Eastern Canada.
Huw received his doctorate in structural RNA biology and protein engineering from the University of Chicago. Alongside his research, Huw was responsible for mentoring new staff on the use of new instruments, including mosquito, where he gained first-hand experience of its reliability and capabilities.
About SPT Labtech
SPT Labtech designs and manufactures robust, reliable and easy-to-use solutions for life science. We enable life scientists through collaboration, deep application knowledge, and leading engineering to accelerate research and make a difference together. We offer a portfolio of products within sample management, liquid handling, and multiplexed detection that minimize assay volumes, reduce material handling costs and put the discovery tools back in the hands of the scientist.
At the heart of what we do
Many of our innovations have been born out of the desire to create solutions to existing customer problems; and it’s this ethos that drives SPT Labtech’s R&D efforts. Our strengths come from the trust our customers have with us to develop truly unique, automated technologies to meet their needs. We combine cutting edge science with first-rate engineering to put customers at the heart of everything we do.
A problem-solving state of mind
The substantial breadth of expertise within our company enables us to be involved in the full life cycle of our products from the initial design concept, mechanical and software engineering and prototyping, to final manufacture and sale. These qualities allow us to offer the best possible technical and mechanical support to all the equipment that we supply, hence maintaining excellent client relationships.