Eppendorf North America
Manager, Application Support
As sequencing costs have fallen, the cost of next-generation sequencing (NGS) library preparation has consumed an ever-larger fraction of laboratories’ sequencing dollars. Today, library prep can nearly equal the cost of sequencing for many experiments. An inordinate amount of manual effort is spent creating libraries, and often several libraries must be generated in parallel. Automating library preparation reduces sample-to-sample and human variability, and best of all, processes can run unattended and without intervention.
Customers, of course, know very well the advantages of automation. But making an automated protocol work properly is trickier than it sounds. If you’re used to building NGS libraries manually, here are four variables to consider as you migrate your processes to an automated liquid-handling platform.
1. Ensure thorough resuspension
Library-preparation protocols typically involve a series of magnetic-bead-based binding and elution steps. Naturally, for those steps to work correctly, the beads must be in homogenous suspension. Although it’s easy to resuspend beads by hand, with an automated system you shouldn’t need to. But take care to ensure that magnetic beads are resuspended during each phase of the library preparation. On an automated liquid-handling platform, the beads can aggregate, and if these ‘clumps’ don’t resuspend properly, all subsequent phases—i.e., washing and elution—will fail.
Eppendorf’s epMotion 5075t automated liquid-handling system is a highly accurate and easy to use NGS library-preparation solution. The 5075t optimizes pellet resuspension using two-dimensional vigorous side-to-side movement, which allows for forceful mixing of very small volumes of liquid. Although traditional 3D mixers or vortexers require vessels to be sealed before mixing and centrifuged afterwards to collect droplets above the liquid surface, 2D mixing requires neither of those steps, meaning the protocol can proceed more rapidly and without interruption.
2. Remove all wash buffer
Wash buffer typically is removed while the work plate is nested above a magnet, but if this stack isn’t programmed perfectly the liquid handler tips won’t be positioned properly with respect to the liquid level. Aspirate from too high, and the tip cannot reach the bottom of the well; aspirate from too low, and the tip will form a vacuum because of high pressure against the bottom of the vessel. Both scenarios ultimately leave a small “dead volume” of wash buffer behind, which can inhibit subsequent elution and/or impact library integrity downstream. Consequently, users generally must monitor their automated system and manually intervene to remove any excess material left behind after a wash.
On the epMotion 5075t platform, a spring-loaded magnet enables automatic contact with the bottom of the vessel. With a spring-loaded backing, the tips can make contact with the bottom of the vessel and completely empty the vessel while avoiding the pressure against the well that causes a vacuum to form, improving overall performance.
3. Maximize template recovery
When preparing a library with small amounts of input material, minimizing loss is imperative. Low yield and large DNA elution volumes negatively affect the ability to generate a concentrated sample for NGS techniques. However, all magnetic beads have a recovery threshold, meaning there’s some template that you just can’t recover off the beads.
One thing users can adjust, though, is vessel geometry—or more specifically, the instrument’s handling of that geometry. The ability to maximize elution recovery is governed by such details as how the software defines your labware, how well the software recognizes the vessel geometry and how precisely it can recover small volumes. On the epMotion 5075t, vessel dimensions are carefully measured and tested, for all manufacturers’ vessels, to ensure high performance and uniformity.
4. Optimize reaction incubation
Finally, consider the exquisite sensitivity of sequencing reactions. Minute variations in reagent volumes, flow and temperature can lead to base substitution errors or incomplete extension. Most NGS chemical treatments—e.g., ligation and end repair—require hot incubation to maximize enzyme efficiency. Ideally, all plate wells should be exposed to the same temperature to avoid sample-to-sample variation. For instance, uneven heating could lead to lower ligation or end-repair efficiency in certain wells.
Many of these issues arise in liquid-automated systems when a heat coil is the primary temperature-control device. The epMotion uses Peltier-driven heating and cooling modules (the same ones used in most thermal cyclers), yielding a high degree of homogeneity from well to well. Peltier technology avoids so-called “edge effects,” whereby the optimal temperature is produced on the outer well portion of the plate. Using this technology, the library-preparation system can deliver the same thermal control and uniformity you expect from your PCR machine.
Conclusion
Next-generation sequencing library preparation is traditionally a long and tedious process, but automation can simplify the work considerably. Precision liquid handling improves the accuracy of both passive and active chemistry steps, which is vital for critical enzymatic processes. A decade ago, the market for automated library prep was limited to high-throughput core labs. Today, low-throughput users increasingly are able to achieve the same benefits and accuracy, especially as personal sequencers become more affordable. Automating library prep is one more inevitable step to keep pace with the sheer volumes of data created. With a system like Eppendorf’s epMotion, that technology is more accessible than ever.
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