Library Prep Tools for Lower-Throughput NGS

It’s easy to find research news about über high-throughput next-generation sequencing (NGS) performed in large projects at major research institutions or companies. But what about smaller research labs? Or even medium-sized labs that want to take advantage of all NGS has to offer? No matter the lab size or throughput, all NGS projects begin with NGS library preparation, an important protocol that prepares the DNA for sequencing. Accurate NGS sequencing begins with a high-quality sequencing library, so investing time and energy in the process of optimizing NGS library prep is worthwhile.

Smaller labs can outsource NGS library prep to a service provider. While this is a convenient option, it is often more expensive, with a long turnaround time. Manual preparation of NGS libraries in-house usually takes a full day for DNA samples, and two days for RNA samples; automation can reduce the hands-on time required. This article discusses challenges facing smaller labs interested in using NGS to sequence only a few samples, and tools to support in-house NGS library prep.

Low-throughput NGS presents unique challenges

Small/medium-sized labs typically can’t afford, or don’t run enough samples to justify, the cost of an automated NGS workstation. The alternative, manual library prep, can be successful with the right set of conditions. For labs that perform NGS infrequently or process small sample numbers, low-throughput library prep presents its own unique challenges when done in-house. “For the researcher who runs only a few samples occasionally, the multi-step process of constructing a sequencing library can be complex, tedious, and very challenging to perfect,” says Cissy Jiang, Global Product Manager at Bio-Rad Laboratories, who notes that constructing complex, high-quality sequencing libraries is an art. “Many commercial kits are comprised of a dozen or so tubes and require multiple pipetting steps, each of which poses a potential for technical error to impact the outcome of the experiment.”

Besides the element of human error, manual library prep can be challenging for smaller labs if personnel are unfamiliar with the necessary technical expertise and optimized protocols, especially if performed infrequently. “Library prep is a complicated process requiring training and experience,” says Ashesh Saraiya, Associate Director of Product Management at Tecan. “Errors during in-house library prep can result in failed libraries, which require repeating the process or even repeating the whole experiment, resulting in project delays and additional cost.”

Fiona Stewart, NGS Portfolio Manager at NEB, agrees that scientists in small/medium labs can be less familiar with the required NGS library prep protocols, which can be overcome with education. “It’s important to utilize clearly written protocols,” she says. “NEB offers many short explanatory videos that cover key parts of workflows and provide reassurance that the user is performing them correctly.”

A particular challenge is the higher per-sample cost. Many library prep kits are designed to process a set number of samples (e.g., 16, 24, or 96) and may include reagents that expire over time. If a lab only needs to run a few samples, the cost per sample will be higher if the remainder of the set of reagents goes unused. Some—but not all—kits allow researchers to split sets and save unused reagents for later. “If the lab chooses to wait and collect more samples in order to run the assay efficiently [as a full set], then turnaround time is impacted,” says Laura Shapiro, Staff Product Marketing Manager for Library Prep at Illumina.

Library prep kits with large sample sets or batches are typically designed for streamlining high-throughput workflows, but some kits are more amenable to low-throughput needs. Look for kits with a batch size that is closest to your lab’s needs, perhaps 8 or 16. “The ability to obtain kits with a low number of reactions enables them to eliminate costs associated with kit waste,” says Ricky Myren, Product Marketing Manager for Liquid Handling at Revvity.

If possible, avoid kits that don’t support the ability to use a small number of samples and save unused reagents. “High-throughput kits may not be developed to support several smaller low-throughput library prep setups—whether due to the consumable configuration, or the reagent stability through freeze thaws, or simply consuming the kit by expiration date,” says Andrew Hutchison, Associate Director of Product Management, Clinical Sequencing and Oncology at Thermo Fisher Scientific.

Designating one person in the lab to keep tabs on the kit components and reagents present in-house is also smart, especially when splitting kit reagents. “Maintaining awareness around kit inventory and expiration date management remains crucial,” says Betsy Young, NGS Product Marketing Manager at NEB, who also suggests looking for reagents with a longer shelf life. “Identifying products that are streamlined for ease-of-use to help minimize the risk of errors is also key, including products that require minimal or no optimization,” she adds.

Look for elements of flexibility

The overall workflow for NGS library prep is basically the same whether performed manually or automated, but kits with more flexibility can make manual prep easier. “For manual library preparation, consider a workflow that has fewer tubes, which typically means fewer pipetting steps and less risk of technical errors resulting in failed library preps,” says Jiang. “Choose a kit that is flexible with a broad input range, so that the user can spend less time normalizing their samples to achieve an optimal condition.”

Another factor to consider when choosing a library prep kit for low-throughput use is the kit’s recommended protocol, as some can be more flexible when it comes to smaller numbers of samples or splitting batches. Particularly valuable for small labs are kits with scalable workflows that can be performed at any level of throughput. “For optimization, the library prep protocols should provide throughput flexibility, allowing for partial LP utilization, and multiple aliquots to be run on different run days,” says Shapiro. “The protocol should specify input size ranges, allowing for variable sample volumes and the automation protocol should have variable throughputs specifically designed for low-throughput use.”

Although most library prep protocols use the same basic steps, some kits include unique components that help to streamline the overall protocol. “Our SEQuoia products feature a novel enzyme that couples the cDNA synthesis and adapter addition into one continued synthesis reaction,” says Jiang. Additionally, Illumina’s bead-linked transposome tagmentation technology provides “a more uniform reaction and reduced library prep time,” Shapiro says, to support fewer protocol steps, less hands-on time, and a faster turnaround.

Adding liquid-handling robots

Even some degree of automation can reduce errors and speed turnaround time. “Lab directors should seek out automation for library prep to streamline workflows and remove as much room for error as possible,” says Hutchison. “Over the long run, automation pays for itself in the reduced labor hours needed compared to manual library preparations.”

Many small/medium labs can benefit greatly by using a liquid-handling robot to automate the repetitive steps most prone to human errors, such as pipetting. “There have been recent advancements in automation to support smaller labs,” says Myren. “It relieves technicians and scientists from manual setups, giving them more time for analysis and yielding consistent data even from inexperienced users.” A wide range of robotic systems are available in different sizes, capacities, automation levels, and costs. An additional benefit of this approach is having a wide range of kits from which to choose (compared to automated workstations).

With regular use, the cost of automating can be recouped due to savings in reagents and time, and the prevention of repeat experiments, but consider this investment carefully. “The cost to purchase and maintain a standard liquid-handling automation system for library prep can be prohibitive for most low-throughput labs,” says Saraiya. “Additionally, programming new scripts, validating workflows, and monitoring the automation system requires staff with the necessary training and expertise.”

It’s important to make a distinction between “automated” and “high-throughput”— automation is not the exclusive domain of high-throughput applications, and has benefits that transcend speed. “The true benefit of automation is the ability to standardize your process and eliminate manual pipetting, which is boring, tedious, and error-prone,” says Jiang. Even without the fastest speeds, a liquid-handling robot offers increased consistency, reduced errors, and more hands-free time compared to manual prep. Library prep kit manufacturers are beginning to offer automated protocols compatible with liquid-handling robot models of varying throughputs. “You may not need to automate all steps of the NGS library prep process to get plenty of value from automation, because even a partially automated process on a low-cost, simple liquid handler can be useful, even life-changing, for small/medium labs running a few samples at a time,” says Jiang.

Fully automated workstations

Fully automated, dedicated workstations for NGS library prep offer the greatest convenience, yet also the highest upfront cost. For example, Thermo Fisher Scientific offers several instruments for automating NGS library prep. “The Ion Torrent™ Genexus™ Integrated Sequencer automates the entire NGS workflow from library preparation to sequencing and analysis all in one run,” notes Hutchison. “The Ion Chef™ instrument provides library automation capabilities for the Ion GeneStudio S5 Systems.”

Manufacturers are beginning to offer benchtop workstations designed to be an affordable solution for automated NGS library prep in smaller labs. “Having pre-developed automated solutions tailored to low-throughput customers is a great solution,” says Myren. “The recently released BioQule™ NGS System is an affordable automated option with pre-developed assays, up to 8 sample throughput, and costs comparable to manual setups.”

Tecan also recently launched the MagicPrep™ NGS system that processes 8 samples per run using cartridge-based reagents with only 10 minutes of set-up time. “The MagicPrep NGS system is designed to be simple to use, involve no scripting or protocol validation, require minimal training, and be highly reliable to specifically address the needs of low-/mid-throughput labs,” says Saraiya. “It provides a new option for fully automated in-house library prep for low/mid-throughput labs and eliminates the burden of manual library prep.”

Despite the benefits of fully automated systems, many small labs still cannot afford such an investment, even taking the long view. In addition, these systems—so far—restrict users to their own compatible library prep kits, of which there is little variety available. However, this may change as the costs of NGS keep decreasing, making it more accessible to a wider range of research labs of all sizes and throughput levels.