Next-generation sequencing (NGS) library preparation is the first step in the NGS workflow. Steps are taken to transform sample DNA or RNA into small nucleic acid fragments, with adapter sequences attached, which are readable by sequencing instruments. To find out more about NGS library preparation, check out The Sequencing Buyer’s Guide.
Historically, NGS library preparation was a time intensive and laborious process. But now, improvements are being made, with many companies offering kits that streamline the procedures, many of which do so by incorporating automation.
Why optimize sample preparation?
The reliability of sequencing data is largely dependent on the quality of NGS libraries. Poor sample preparation produces skewed and unreliable sequencing data, ultimately compromising the research. Although the composition of samples is highly important, library preparation procedures are crucial for maximizing and maintaining the quality of nucleic acid mixtures. Moreover, optimizing these processes ensures a consistent yield across samples, in turn making sure that the DNA is accurately represented in the sequencing flow cells.
Why automate NGS library preparation?
Many scientists use manual workflows for NGS library preparation. However, this can lead to significant disadvantages because the procedures often have numerous and repetitive steps that require high precision pipetting. This can introduce variability between users, even with experienced scientists. Small differences in pipetting techniques can be amplified further down the workflow, causing considerable inconsistencies in the library yield and quality. Furthermore, during manual workflows there are multiple opportunities for the contamination of samples, in turn causing errors and hindering the reliability of the sequencing data.
Automating NGS library preparation has the potential to address these issues. It is an effective strategy for reducing the number of manual stages required and promotes a simpler workflow with fewer pipetting steps.
Automated library preparation advantages
- Faster
- More efficient
- Less prone to human errors
- Less susceptible to contamination
- More reliable and consistent
NEBNext are a line of automation-compatible NGS library kits, marketed by New England BioLabs. The products support Illumina sequencing with library preparation tools that streamline workflows and reduce hands-on time. Agilent also offers kits for automated NGS library preparation, called the SureSelect Low Input Kits. There are XT, XT2 and HS versions available, depending on what sequencing platform is being used. The Magnis Prep System, which is compatible with the HS kit, is a benchtop instrument that handles intact or degraded FFPE samples, incorporates automated barcode checking and carries out self-checking routines.
One of the easiest methods to incorporate automation in this step of workflow is with magnetic beads. This is because only a multi-well plate format and a magnetic plate are required for the systems to be used. NucleoMag NGS Clean-up and Size Select is a tool for fragment size selection and reaction clean-up, whereby beads selectively bind to DNA fragments, in turn purifying the library of any contaminants (primers, adapters or enzymes). The kit can be used either manually or through automating on standard liquid handling instruments.
Drawbacks of automated library preparation
Despite it becoming increasingly popular to automate library preparation, some systems still require a significant amount of hands-on time due to user input in reagent preparation and loading. Furthermore, library quality control methods have proven difficult to integrate into automated systems, meaning that many researchers still have to perform these stages manually. Library quality control is an essential component of the NGS workflow as it is necessary to confirm the nucleic acid quality at various checkpoints to ensure successful sequencing outcomes. However, it is also one of the most error-prone stages of library preparation. Therefore, if automated NGS workflows are unable to include library quality control, they are still prone to concentration variation.
However, recently, fully automated systems are being introduced, providing innovative new solutions to help improve NGS library quality whilst transforming workflow efficiencies. For example, the NGS DreamPrep provides complete NGS library preparation automation, including a novel library quality control method, called NuQuant. The system and kit are marketed by Tecan, a global provider of automate laboratory instruments. Their automated platform was the first to fully integrate library quality control and libraries for DNA sequencing can be prepared in less than four hours.
Fully automated systems, such as these, have the potential to advance genomics research and generate robust NGS data. These library preparation systems also save a great deal of time, reducing the total duration of workflows and dramatically increase laboratory productivity. Therefore, more time can be dedicated to other projects and help to accelerate potential research.
Tips for digital NGS library preparation
Nevertheless, there are a number of considerations that need to be taken before fully automating NGS library preparation. The use of a closed platform, tied to a single supplier, may be cheaper initially, but consumables can become more expensive over time. Choosing an open platform can combine the best solutions from a diverse set of suppliers, and can be easier to adapt following a shift in workflow protocol. Moreover, it is recommended that researchers think about sample numbers when switching to automation because each system has different processing capabilities. The level of desired automation should also be considered. For example, if a laboratory is looking for a fully automated workflow, a system should be chosen that has a ‘touch button walk-away’ ability.
Once companies automate library preparation, it is important to consider how best to optimise the protocols. Perhaps the most effective way of doing this is performing quality control at several steps throughout the preparation. For example, tools are available for the automated electrophoresis of nucleic acids, such as the Fragment Analyzer, that can check raw material and score the DNA according to its quality.
Image credit: Scientist Live
