Reaction Miniaturization for NGS Library Preparation

Reaction minimization is a developing trend within the field of genomics, particularly in library preparations for next-generation sequencing (NGS). Scaling down reaction volumes for this process comes with numerous benefits, which are further enhanced with the integration of automation systems. In this article, two automation experts share their insights into the benefits, challenges, and implementation advice for reaction miniaturization in NGS library preps.

Understanding reaction miniaturization

"Reaction miniaturization is the process by which a particular reaction step in an NGS workflow, or any other biochemical reaction, is optimized for lower reaction volumes,” explained John Fuller, Ph.D., Commercial Product Manager for Echo Instruments at Beckman Coulter Life Sciences. He noted this optimization can involve simply dividing the reaction components and observing their performance, or it may require more specific adjustments to certain elements of the reaction to ensure efficient miniaturization.

“The concept of miniaturization isn’t new,” noted Kamila Koprowska, Ph.D., Senior Business Development Scientist at SPT Labtech. In fact, for many years scientists have been manually scaling down their reactions to extend the use of reagents and samples. However, Koprowska pointed out that automated liquid handling technologies have opened up the possibility for more aggressive miniaturization. Koprowska further shared that miniaturization within NGS library preps has gained traction because it helps tackle major challenges in genomics such as rising research and development expenses, greater demands for throughput, and the requirement for consistent and dependable results.

Advantages of miniaturization

One of the most obvious benefits of reaction miniaturization is that the reduced volume of reaction mixtures leads to decreased consumption of expensive reagents and valuable samples. Koprowska noted that these cost savings become more substantial as labs increase sample throughput with high-throughput sequencing systems. “In our experience, miniaturization can reduce experimental costs at least by 75% while preserving the same success rate and method sensitivity,” she stated. Aside from cost savings, Koprowska highlighted that miniaturization improves the resolution of genomic and NGS libraries by increasing sensitivity. For instance, the use of smaller volumes in research like single-cell studies enhances resolution, allowing for the detection of a greater number of genes and more detailed genetic insights.

With the advent of new technologies, laboratories are equipped to handle more sequencing samples, but they are constrained by the tedious nature of sample and library preparation, which can restrict progress. Koprowska noted that this is especially true for large-scale studies like single-cell analysis in spatial biology, pathogen surveillance, population genomics, and proteomics using NGS readouts. These processes require extensive time and labor efforts and also lead to considerable expenses in library generation. A major advantage of miniaturizing NGS library preparations with automated solutions is that it addresses significant scaling challenges in sample preparation. Fuller explained that this increase in throughput allows more samples to be sequenced, leading to robust data through replication. Additionally, miniaturizing NGS reactions through this process allows for the generation of additional data points, which helps create more detailed models and increases reliability through improved reproducibility.

Implementation advice

Researchers looking to implement reaction miniaturization in their own NGS workflows may face several initial challenges. “The first challenge is having a thorough understanding of the underlying chemistry of the reaction and the end goal,” emphasized Fuller. This also includes knowing the specific degree of miniaturization desired and carefully selecting the appropriate tools for the process. Fuller shared that scientists typically address these problems by employing a design of experiments (DoE) approach to determine essential conditions like cofactor concentrations for reproducible results. In addition, automation plays an important role in standardizing these processes, which is often done in collaboration with automation experts to further refine the workflow.

Koprowska cautioned that as reaction volumes decrease, pipetting errors, influenced by factors such as air pressure or viscous and volatile reagents, become more significant. This can lead to pronounced errors in smaller volumes, where a slight variance can substantially impact the reaction. Fortunately, Koprowska emphasized that with enhanced pipetting accuracy, automated liquid handling systems can facilitate reductions in volume to 1/50^th of the original. In particular, Koprowska recommended utilizing systems with positive displacement technology, a method where the piston comes into direct contact with the liquid, to maintain accuracy across all types of liquids. This ensures accuracy with all liquid types and supports confident work with nanoliter volumes for further miniaturization.

Along with these considerations, Koprowska suggested striking a balance between cost savings and assay sensitivity. “As NGS is such a dynamic field, opt for a solution that offers flexibility,” she added. Adopting an intuitive, easy-to-use platform allows for the integration of emerging chemistries, as well as enhancing lab efficiency and resource use. Koprowska explained how this simplifies training, encourages widespread adoption among teams, and reduces the risk of underutilization if expert users leave the lab.

In addition, Fuller suggested having a detailed conversation with the local team of automation representatives, project managers, and application specialists on anticipated goals for the coming 6–12 months. This includes establishing specific weekly or monthly throughput targets and addressing any existing constraints and bottlenecks that might create challenges. “Lastly, I’d advise coming together as a group with a fresh look at newer chemistries or technologies that might enable a complete shift in how the workflow performs,” Fuller recommended.

Innovations and future prospects

Recently, Fuller has engaged with several vendors in collaborative efforts to optimize reaction automation and miniaturization for research. This includes bringing scalable sequencing to burgeoning applications such as plant and animal sequencing and other targeted genotyping applications. Fascinated yet inundated by the rapid development of new chemistries and emerging technologies, Fuller sees a future where NGS library prep automation and miniaturization co-evolve in intriguing ways. He also anticipates greater integration of reaction miniaturization with multiomics-based methods. Emphasizing the potential of these advancements, Fuller stated, “Being able to capture nucleic acid sequences along with the proteome and metabolome of samples in a scalable manner (and being able to make intelligent inferences with that data) is something that will take life sciences to the next level, and it’s thrilling to have a front-row seat.”

As for Koprowska, she foresees miniaturization and automation playing an important role in advancing genomic sequencing toward personalized medicine. Despite its potential, there are many obstacles on the way to routine practice, particularly in understanding health and disease in underrepresented populations. Efforts to bridge this gap include supporting large-scale studies and enhancing genomics training in underrepresented regions. Koprowska also believes that user-friendly automation and efficient reduction of reaction volumes will help to address these challenges. A prime example is the U.K.’s New Variant Assessment Platform (NVAP), which supports 18 countries and 6 regions and provides automated liquid handling technology to enhance genomic sequencing capabilities globally.