GENERATING A HIGH- QUALITY LIBRARY FOR NEXT GENERATION SEQUENCING THROUGH AUTOMATION
Using automation for library preparation can provide higher precision and reproducibility
Next generation sequencing (NGS) library preparation workflows vary widely but share common challenges stemming from their sensitivity, requiring precision pipetting in both volume and time while handling ethanol, beads, and viscous reagents.
Library quality is critical to success, and the time and expense that go into prepping a sequencing run add pressure to get it right the first time every time. Reproducibility, however, is difficult to achieve with manual pipetting. Library preparation workflows consist of a large number of pipetting steps, making them prone to human error, exhausting for lab technicians, and a source of risk for repetitive strain injuries. They also have numerous timed steps and require pipetting small volumes, which are difficult to complete consistently across a plate. Small differences add up quickly and can have a measurable impact on sequencing results.
Many labs solve these precision challenges using automated liquid handlers, but the typical large platforms have a high entry-point and are not practical for many smaller labs. These platforms also require considerable training for set up and use, with some requiring coding experience and others that are programmed to only run a specific protocol that can be difficult to edit. A smaller, more flexible system is better suited for running different types of methods, adapting to changing lab needs.
Entry-level liquid handlers that automate key pi- petting steps offer an ideal solution for labs transitioning from manual library preparation. These instruments are typically much easier to use and effectively streamline the process. While some hands- on time is required, this level of automation increases batch or experiment-to-experiment consistency and reproducibility, reduces hands-on time, and per- forms time-critical steps with precision.
For best results when making the transition, look for platforms with optimized and validated NGS protocols. For example, the epMotion® series of liquid handlers from Eppendorf have automated proto- cols for prep kits from all of the major manufacturers qualified and available via application implementation for use with optimization, customization, and training support. Validated protocols on such plat- forms save labs considerable time and effort when transitioning to automation while boosting confidence in results. The benefits of automation, including reduced human error, increased pipetting precision, and improved execution of timed steps, can dramatically increase NGS library quality.
MINIMIZING ERROR
A number of easy-to-make mistakes while manually prepping libraries can nullify results. Common—but major—mistakes include missing adapters in some tubes or wells, adding the same barcode to multiple samples, cross-contaminating or mixing up samples, or missing or double-loading a re- agent into a well or tube. It is also easy to aspirate sample while on the magnet, through imprecise tip placement or timing.
Automation minimizes these types of errors, which are often driven by fatigue or excessive workloads, though there is still room for human error. One potential problem introduced with automation is set- ting the deck incorrectly, which can result in crashes or otherwise failed routines. More subtle errors can be introduced during programming.
Smaller, less complex liquid handler platforms can offer a great entry point, typically being much easier to set up, customize, and use. Some platforms further minimize errors and program requirements—for example, the epMotion dynamically switches between tools to optimize run time and reagent usage. It also performs an optical scan before each run to verify correct labware, vessel placement, lid opening, and liquid levels on the deck. Available NGS methods are qualified by kit manufacturers and customers to meet a data quality level of performance agreed upon by both parties to produce high quality libraries. Customizing programs is easy—the intuitive, drag-and-drop based epBlue software allows users with no coding experience to quickly modify existing methods or program new methods from scratch, but on-demand method development services by ap- plication specialists are also available whether for small adjustments of existing protocols or entirely new or custom workflows.
BOOSTING PIPETTING PRECISION AND ACCURACY
Precision and accuracy during manual pipetting are an ongoing concern in the lab for most proto- cols. It is particularly critical for NGS runs, how- ever, where the quality and repeatability of results rely on consistent depth and complexity of resulting sequence libraries. Biases are magnified during PCR and normalization stages. Normalization helps ensure that samples are equally represented in the pool and must be performed with a high degree of precision.
Many protocols use beads for normalization, tag- mentation, and/or reaction cleanup. Bead protocols are highly sensitive to ratios and require precision while pipetting challenging liquids like ethanol and beads. Small changes in bead-to-sample ratios are difficult to avoid when manually pipetting small volumes and will skew the fragment length distribution within the library.
Beads are frequently used for purification and enrichment—they bind DNA and are rinsed with ethanol to remove dimers and other short DNA sequences. With each rinse, the plates or tubes are placed on a magnetic stand to pellet the beads, allowing liquid to be replaced. It is imperative to avoid removing portions of the sample by using careful and precise movements in placing the tip of the pipette to bypass bead clusters and by not re- moving liquid until the beads have had adequate time to migrate to the magnets.
Reactions across the board rely on precise and accurate pipetting, as receiving even slightly different quantities of reagents will alter the efficiency of the reaction, which can skew results. This is particularly critical when setting up amplification and tagmentation reactions.
Automated liquid handlers save the day on the pipet- ting precision front as well, for both viscosity and volume. Instruments with multiple calibration points maximize precision for high viscosity liquids. Some, like the epMotion® platforms, offer improved pipetting of volatile reagents like ethanol and other challenging liquids. The epMotion is also capable of accuracy and precision down to 200 nL, supporting sample volume miniaturization.
IMPROVING TIME-CRITICAL STEPS
NGS library preparation protocols contain multiple timed steps where a lack of precision will negatively impact results and repeatability.
Fragmentation and tagmentation
DNA and cDNA are typically fragmented, blunt- ed and phosphorylated, A-tailed, and ligated to adapters early in the library prep workflows. Frag- mentation, whether enzymatic or physical, requires precision to generate target fragment lengths consistently across samples to form comparable libraries. For enzymatic reactions, fragment size is largely dependent on reaction time. Longer reaction times give a higher proportion of small fragments, effectively altering the sequencing library.
Some library preparation kits use a “tagmentation” step that fragments, repairs ends, and ligates simultaneously using transposons or enzymatic beads, reducing sample handling and saving time. This step is sensitive to both amount of input DNA and re- action parameters, with timing again being critical.
Bead washes
Magnetic bead wash steps, in addition to presenting challenges around pipetting precision, are sensitive to timing. Between ethanol rinses and elution into a buffer for downstream reactions, a timed drying step is used to remove the remaining ethanol. If the sample is
not adequately dried, ethanol can be carried forward and inhibit downstream enzymatic reactions like the PCR reaction. Too much time at this step will overdry the beads, making the DNA/bead complex hydrophobic and preventing elution.
Enzymatic bead protocols for tagmentation move through wash steps with similar constraints, also at risk of substantially lower yields if dried.
Automation can boost performance by executing delicate protocol steps with more precise timing.
cDNA synthesis
RNA sequencing enables numerous tools for path- way analysis and target identification and can be used to detect changes to structure or quantitatively analyze gene expression when interrogating novel features or those with a low background signal.
Working with RNA brings additional challenges, however, being prone to degradation with contamination forming a major concern. RNA must be kept on ice with minimal freeze/thaws cycles and early steps must be performed quickly to achieve the best results.
Article designed by
Automated liquid handling for these steps can ensure both precision across all samples in a plate, regardless of position, and minimal contamination risk. Some liquid handlers, like the epMotion®, provide an integrated thermal module on deck for reliable temperature control throughout the process.
NGS library preparation protocols are sensitive and critical to get right. Frequently, technicians that are accustomed to manual library preparation are hesitant to trust the delicate workflows to automated plat- forms, but transitioning to automated workflows with the right platform and support can drastically improve sequencing data. Automated liquid handler platforms help eliminate the risk of human pipetting errors, in- crease productivity, improve repeatability and accuracy, and can help maximize reaction efficiencies and yield through precise management of timed protocols. Eppendorf offers reliable platforms, accessories and services that smooth the transition to workflow automation, providing the same level of quality customers have come to expect since launching the first commercial piston pipette in 1961.
