DNA sequencing was an enormous scientific breakthrough that enabled investigation into various biological processes pertaining to human disease, genetic inheritance, immunity, and cancer, among countless others. The first rapid DNA sequencing technique was developed by Nobel Laureate Frederick Sanger and his colleagues in 1977. The chain termination method, now termed Sanger Sequencing, relies on a DNA polymerase to produce copies of a single stranded DNA template. Labeled deoxynucleotides are incorporated and the resulting DNA fragments of various lengths are electrophoresed and laser excitation is used to read the sequence and produce a chromatogram. While the Sanger technique was widely used, it has several weaknesses including low throughput, scalability, cost, speed, and resolution. More recently, the development of next-generation sequencing (NGS) has markedly improved on the limitations of Sanger sequencing. NGS enables millions of sequencing reactions simultaneously, reducing the cost and increasing the speed of DNA sequencing. NGS depends on a high-quality library prepared from genomic DNA and RNA, a process that involves copious amounts of pipetting and attention to detail. Such tasks may consume large portions of a working day that would otherwise be spent writing papers and grant applications, or staying up to date on new literature. Innovative automated solutions simplify NGS library preparation, increase productivity, and eliminate time lost to tedious tasks such as pipetting.
Precise and accurate liquid handling for better data quality
NGS library preparation requires DNA fragmentation and addition of adapter sequences, polymerase chain reaction (PCR) methods for amplification, followed by gel purification. The process is labor-intensive, mainly due to the enormous amount of time spent pipetting. There is also a high risk of error when pipetting manually due to inconsistent technique, pipetting into the wrong well, and even skipping wells. Automating the process saves the operator countless hours of bench work and improves accuracy and precision to ensure high quality data and prevent costly errors.
Well-designed automation software can automate a pipetting protocol into a ready-to-run procedure and accounts for dispensing speed, aspiration, and blow-out parameters based on liquid characteristics. More viscous liquids, for example, should be dispensed slowly compared to more aqueous liquids and a longer blow delay will allow the liquid to flow down the tip completely. An automated alternative, such as an Eppendorf epMotion NGS solution offers settings for different liquid types, and the ability to modify individual parameters to ensure optimal liquid dispensing. Kits for NGS library preparation workflows are also available, and have been qualified by reagent manufacturers to ensure accurate results. Software should also be intuitive and easy to use, so that multiple users are able to operate the system implementing their own methods. For NGS applications, hiring a specialist to develop a method can save time and expense spent optimizing hundreds of different steps.
For laboratories that need to process multiple different library preparation workflows, flexible open platform instrumentation is essential. Automated solutions with multiple deck positions, gripper options, and automatic exchange between single or multi-channel dispensing, and 96 or 384 well plates are suitable for a wide range of workflows.
It is critical to avoid contamination during library preparation, especially during PCR processes. This often requires separating pre- and post-PCR processes spatially, and working with great care and attention to detail. It is recommended that pipette tips be changed between each sample, and between each row and each column when adding adapters. Automated Eppendorf systems offer contact-free dispensing, eliminating any potential cross contamination between liquids and samples and prevent user errors such as neglecting to change a pipette tip. Prior to a run, the automated epMotion system checks the deck to ensure the appropriate type and number of pipette tips are present, and reliably performs otherwise tedious pipetting tasks.
Automated systems may be equipped with ultraviolet lights and HEPA air filters to ensure sterility and prevent contamination. It is also important to work with low binding consumables to prevent sample loss due to adsorption to the plastic.
Magnetic separation, mixing, and temperature control
Bead-purification steps and temperature controlled enzymatic incubations are part of the DNA and RNA purification process for NGS workflows. For optimal results, powerful magnets, variable mixing speeds, and precise temperature control are required. epMotion technology incorporates magnetic fingers that draw in and out of the sample rack to facilitate mixing, washing, and separation of magnetic beads. Mixing frequency has been optimized for specific labware and applications, and the system delivers stable temperature with precise control. It is also essential to avoid disturbing the magbead pellet, which is a risk associated with manual pipetting, especially when working with a multichannel pipette. Automated pipetting eliminates this risk and saves time compared to careful manual pipetting.
In addition to hours spent pipetting, the combination of bead purification steps and temperature controlled enzymatic incubations necessary for NGS library preparation can leave the operator feeling tethered to the bench for hours at a time. A reliable automated system provides the peace of mind necessary for the operator to step away from the bench during the process.
Automated solutions eliminate bottlenecks
NGS systems continue to evolve, offering greater throughput, shorter run times, and better analytical techniques. Improved technology is matched with growing demand for fast answers, more affordable sample analysis, and better data. NGS library preparation remains the bottleneck between new technology and research demands, as it is a specialized, time-consuming process crucial to ensure data quality. Automated solutions can eliminate this bottleneck, as they perform otherwise tedious and time-consuming tasks with high accuracy and precision. When investing in an automated system, it is important to select a reliable, industry-proven manufacturer such as Eppendorf.