Why Automation with the Correct Liquid Handler is Integral to Improved NGS Outcomes

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Pharmaceutical companies are under constant pressure to develop and get approval for life-saving drugs. As the race intensifies, the complexity of novel therapeutics and the speed of delivery to market must increase concurrently.

So far, 2023 has been a productive year as 29 new drugs have been approved by FDA for various conditions, including heart failure, pneumonia, B-cell lymphoma, and migraine, as well as rare genetic disorders, such as Rett syndrome and Friedreich ataxia (1). In Europe, the European Medicines Agency (EMA) issued marketing authorization for 89 medicines in 2022 (2). In particular, oncology treatment was at the forefront with the approval of eight drugs by the EMA in the first few months of 2024.

Getting to this point has not been easy for the bio/pharmaceutical industry, given that the average time from initial discovery through approval can reach 10–15 years. Nevertheless, the COVID-19 pandemic showed that drug product timeline can be accelerated significantly in the face of an urgent global threat. Automation in early drug development can help address a host of challenges, as manufacturers require consistency and timeliness in drug production.

Next-generation sequencing in patient stratification and drug development

Many groundbreaking discoveries in the pharmaceutical industry would not be possible without high-throughput technologies that allowed for the evaluation of a large number of samples at pace. In particular, next-generation sequencing (NGS) has revolutionized the landscape of drug discovery. Thanks to this massively parallel sequencing technology, scientists can rapidly generate a complete library of DNA and RNA fragments from whole genomes, transcriptomes, and related sequences (e.g., epigenome). NGS has been used widely to study the human microbiome, sequence cancerous samples for genetic variants, and analyze the molecular mechanism of action of small molecules.

Whole genome sequencing makes possible the comparison between different genomic sequences, allowing researchers to identify disease-associated mutations, which essentially drives target discovery. Furthermore, potential response to drug treatment can be assessed by investigating changes in the transcriptome upon testing with cell and organoid-based disease models. Additionally, providing deeper insight into the drug mechanism of action, NGS can help identify potential off-target effects early in R&D. The role of NGS is even more evident in cell and gene therapies employing complex genome editing mechanisms. When developing such therapeutics, researchers need to ensure that the genome editor is acting precisely on the target site and not causing off-target modifications.

As understanding of the human genome evolves, the pharmaceutical industry is shifting from a generic-drug product for large patient populations to more tailored treatments for subpopulations according to their specific genomic profiles. In this regard, NGS is paramount to stratification efforts because it can uncover not only common mutations but also previously unknown subgroup-specific mutations. By stratifying patient profiles, researchers can better predict the outcomes of drug treatments and identify treatment-resistant groups requiring more advanced therapies.

Library preparation: the main challenge in NGS workflows

NGS workflows are fairly straightforward on paper, consisting of the library preparation, sequencing, and data analysis steps. To add to that, relevant instrumentation has improved swiftly since the first NGS launch in 2000 (3), bringing improved ease of use, reduced costs, and high-throughput sequencing capabilities. Nonetheless, library preparation remains a bottleneck that can derail sequencing data generation.

Obtaining high-quality samples to prepare large libraries of DNA fragments can be difficult to afford due to factors, including limited annual budgets and increasing sample costs. One way to mitigate costs is the reduction of sample volumes; however, with manual pipetting still being a prominent method in research laboratories, this is not an easy task. Even with the involvement of highly trained staff, manual library preparation is still prone to human errors. Manual tasks performed at different times and/or by different staff can lead to well-to-well variability in volume and sample quality. Moreover, the high load of sequencing data generated from manually prepared libraries can be arduous to organize and analyze. More importantly, volume reduction may not be sufficient with manual pipetting because the minimum required DNA volume is still in the µL scale. With these factors combined, cost reduction is substantially constrained when using manual workflows.

The setbacks of manual library preparation can be alleviated by automating liquid handling such that minuscule amounts of DNA can be dispensed into microplate wells to construct libraries with unmatched precision in a short time frame. There are different types of liquid handlers with distinct advantages and precise liquid ejection capabilities. Currently, a tip-based liquid handler is the most common technology used for NGS automation, where the liquid is ejected from a plastic tip via contact dispensing; however, precise dispensation can be hindered by damaged tips. In addition, possible interactions between the reagent and the plastic tip may cause variations in the droplet volume.

Acoustic liquid handlers surpass tip-based liquid handlers in precision with their cutting-edge working principle that deploys sound waves to eject nanoliter-scale droplets onto surfaces. Droplet size is pre-determined by setting up the frequency of sound waves. This highly precise technology is contact-free and equipped to handle transfer volumes between 2.5–25 nL. Compared to traditional liquid handling methods, the acoustic transfer capacity can decrease transfer volumes by over 75% (4). Thus, researchers can prepare larger libraries with a fixed annual budget, while maintaining the integrity of delicate DNA fragments in the sample.

Traditional liquid handlers may require manual calibration when working with a wide range of reagents. In contrast, thanks to the ability of acoustic handlers to automatically adjust certain fluid transfer parameters in real-time, researchers gain the flexibility to work with a broad range of complex reagents. The simultaneous normalization feature would particularly benefit users working with high-concentration libraries, as the need for library dilution would be eliminated.

Perhaps the main advantage of acoustic liquid handlers is the ability to build libraries at unprecedented speed. The acoustic droplet ejection technology enables a transfer speed of up to 700 droplets per second. As a result, particular workflow steps such as pooling and normalization that previously took hours with tip-based liquid handlers can be completed in minutes through acoustic liquid handling.

Strategies before deploying automated liquid handling

NGS offers high-throughput genomic analysis and can greatly contribute to target discovery and drug development. The optimum NGS workflow design is a must for achieving actionable insight from sequencing data without straining budgets allocated to R&D. That’s why the choice of liquid handling is of central importance. While the advantages of non-tip-based acoustic liquid handlers are clear, the choice of which to use is not always straightforward. As mentioned before, different types of liquid handlers come with distinct advantages. For instance, tip-based liquid handlers are widely used because of their excellent capabilities of bead cleanup for purification, while acoustic liquid handlers may fall short in that regard.

The main takeaway is that NGS automation projects excel when collaborative subject matter expertise is used to deploy the correct automation solution to solve a particular challenge for sequencing. Tip-based and non-tip-based handlers can be paired together to garner maximum benefit from both types. Nevertheless, merging different liquid handlers can be overwhelming, considering the company’s research needs, timeline, and financial resources. Field experts that manufacture liquid handlers and devise assay workflows can be extremely valuable guides. As the health and life sciences industries make progress with precision medicine, flaws and delays in preclinical research are less easily tolerated. For that reason, companies should not hesitate to consult expert manufacturers to successfully incorporate liquid handling into NGS workflows so that they can generate vast libraries for actionable genomic sequencing and analysis.

Products referenced are not intended or validated for use in the diagnosis of disease or other conditions.

References

1. FDA. Novel Drug Approvals for 2023. www.fda.gov (accessed Sept. 29, 2023).
2. EMA. Human Medicines Highlights 2022. www.ema.europa.eu, 2023.
3. Barba, M.; Czosnek, H.; Hadidi, A. Historical Perspective, Development and Applications of Next-Generation Sequencing on Plant Virology. Viruses 2014, 6 (1), 106–136. DOI: 10.3390/v6010106
4. Beckman Coulter Life Sciences. Australian Lab Achieves High-Throughput Genotyping by Amplicon Sequencing Using Echo 525 Acoustic Liquid Handler and Access Systems. Case Study, beckman.com (accessed Sept. 29, 2023).

About the author

John Fuller, PhD, is commercial product manager for Echo Instruments, Beckman Coulter Life Sciences.