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By Roberta Veneroni
Next-generation sequencing (NGS) is driving dramatic progress in many fields of research. However, the value of NGS data is often limited by factors such as poor analysis pipelines and poor library quality. One way to improve the quality of your libraries is to optimize your NGS library prep, but this can be challenging, as the process involves multiple steps that can introduce user-user variability and the risk of contamination.
Automated NGS library prep has the potential to address these issues. However, many automated systems still require significant hands-on time. This is because they can involve a lot of user input in reagent preparation and loading, and they can’t fully automate the labor-intensive steps for QC. To solve this problem, a fully automated solution with integrated QC has recently been introduced. The system provides an innovative new solution that could help you improve NGS library quality while transforming your workflow efficiencies.
Fully integrated NGS library prep automation could be the key to improving your data quality and efficiency.
Why is it so important to optimize NGS library prep?
If you’re working with NGS, it’s no secret that the reliability of your data is highly dependent on the quality of your libraries. With low-quality libraries, you’ll struggle with skewed and unreliable data that could ultimately compromise your research. While library quality ultimately depends on the composition of your samples, it is also influenced by the nature of your library prep process. Therefore, it’s vital to ensure that your workflow is designed to maximize the quality of the samples you have.
Another major reason to optimize your library prep is to ensure a consistent yield across multiplexed samples. Inconsistent library concentration is a significant concern, as it can result in incorrect equimolar pooling before sequencing. In turn, this can result in the misrepresentation of samples in the sequencing flow cell, which could result in under- or over-sequencing some of the samples in your pool. For example, lower concentration libraries can be under-clustered, so you may not achieve enough depth of sequencing for these samples. As such, you may have to perform re-runs, wasting valuable time and resources.
To overcome these challenges, you need to optimize your NGS library prep. Not only will this increase library quality and allow you to achieve a more consistent library concentration, it will also improve the overall time- and cost-efficiency of your NGS workflow.
The pain points of manual NGS library prep
Many scientists still use manual workflows for NGS library prep. However, this has significant drawbacks because library preparation often has numerous, repetitive steps that require small volumes to be pipetted with high precision. As such, the high user input required in these workflows can compromise library quality and yield for several reasons.
Firstly, the numerous manual steps can introduce variability between users. This is a common problem even with experienced scientists, as small differences in pipetting techniques can result in considerable variability in library yield and quality, meaning it’s not possible to achieve optimal sequencing.
Another disadvantage of manual library prep is that there are multiple opportunities for introducing error and contamination, which will ultimately reduce library quality. In turn, the reliability of your sequencing data will also be compromised, which could hinder the progress of your research.
A simpler workflow with fewer pipetting steps will help you to overcome many of the challenges of producing ready-to-sequence libraries. Automating your processes is an effective strategy for reducing the number of manual stages required, which can go a long way towards solving your library prep challenges. However, there are certain limitations to look for when you’re evaluating automated systems.
What to watch out for in automated NGS library prep systems
Automated library prep systems are becoming increasingly popular, as they have the potential to overcome many drawbacks of manual workflows. However, automating library prep is a complex task and it’s important to note that some systems still involve a substantial amount of manual work.
One reason for this is that some automated systems require significant effort for worktable set-up and for performing incubations offline. What’s more, there can be considerable hands-on time required for reagent preparation, and loading procedures can be complex. All this adds up to significant manual input.
Another major source of user input in automated library prep workflows is QC. Library QC, the process of measuring and normalizing library concentration, is one of the most error-prone stages of library prep. However, it has proved difficult to integrate commonly-available QC methods into automated systems, meaning that researchers have to perform this stage manually. The result? Libraries that are still prone to concentration variation.
Fortunately, a novel QC method, known as NuQuant®, has been developed to address this issue. This method uses a direct fluorometric assay to enable efficient QC at a fraction of the time taken by previous methods. The major advantage here is that the NuQuant method is integrated into select library preparation kits from Tecan, which are optimized to run on automated systems. While a range of other automation-friendly kits have been available for some time, these are the first to incorporate library QC. Therefore, these new kits open up the potential for full NGS library prep and QC automation.
Full NGS library prep and QC automation: The key to optimizing your workflow
Complete NGS library prep automation, including library QC, now has the potential to fully optimize your library prep workflow. A new system, the NGS DreamPrep™, provides the first automated solution able to deliver quantified and normalized DNA or RNA libraries ready for pooling. In this way, the fully integrated solution delivers a number of advantages over previously available automated platforms.
Firstly, with fewer manual stages than any previous system, this workflow has minimal potential for error and user-user variability. Therefore, provided your samples are of adequate quality, you will be able to produce consistently high-quality libraries. As such, you will be able to generate reproducible and reliable NGS data. With consistently high-quality NGS data, you can make the most of your samples and advance the potential of your research.
Cutting down on manual stages will also free up your time, which you can dedicate to other projects. If routine and repetitive manual stages are automated, you are free to invest your time in doing science, helping you advance your research.
As well as reducing the manual work you need to put in, the NGS DreamPrep solution can free up even more of your time by reducing the total duration of your library prep process. In fact, the time savings are quite incredible – for DNA sequencing, libraries can be fully prepared and quantified in less than four hours, compared to seven hours for other systems. Much of this time saving comes from using simple kits with integrated QC, which are designed to work optimally in combination with the advanced hardware. Since it’s possible to nearly halve your time investment in library preparation, this fully integrated workflow could make a dramatic difference to your lab productivity and accelerate your research!
What’s more, with this new solution you won’t suffer from sample loss in QC, as the output library plate can be read directly with any plate reader. Therefore, you can preserve precious library material, enabling you to get more value out of each sample.
Finally, having a fully integrated system can make it much easier to troubleshoot problems. This is because it’s possible to use the same supplier for the entire workflow, so you only have to look to a single source for support.
The benefits of optimizing your workflow with automated NGS library prep
NGS library prep automation has great potential to advance your genomics research by helping you to improve your library quality and achieve consistent yields. As such, you can be confident that your libraries will help generate robust and reliable NGS data. In addition to improving the quality of your data, NGS library prep automation can also enhance your time- and cost-efficiencies, helping you to accelerate your research.
While many automated systems still require significant user input, particularly in the QC stage, the NGS DreamPrep now provides a fully integrated system to streamline and optimize your entire library prep process. With this system, you can ensure you gain the maximal possible benefit from automation.
To learn more about the NGS DreamPrep and discuss how to bring its advantages to your own lab, book a meeting with one of our experts.
About the author
Evaluating the quality of DNA for Next Generation Sequencing, genotyping, and other downstream applications