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By David Wold
With high-throughput genomics impacting every corner of biology, the demand for more efficient Next-generation sequencing (NGS) workflows is growing rapidly. Automating the process of NGS sample preparation is crucial to avoid inaccuracies due to human error, bottlenecks that delay sequencing results, and the additional expense of re-running sequences. What are the most important factors for an engineer to consider when selecting a pump to meet the stringent performance required for an automated NGS library preparation system?
First, a very short background in NGS sample preparation
The first step in preparing samples for NGS is to extract and purify DNA or RNA from a biological sample. This step is commonly called sample preparation. The next step is called library preparation, it involves cutting the DNA or RNA of interest into fragments and then selecting specific fragments to be sequenced. The resulting collection of ready-to-sequence fragments is called a library. In this blog, I use the term “sample prep” to encompass the entire process of preparing sequencing-ready samples for NGS.
Pump precision and accuracy are essential for high-quality NGS library prep
NGS itself is a very high-throughput process, so automating the upstream sample prep workflow is essential to enable fast and efficient parallel processing with minimal manual intervention. Compared to other high-throughput genomic analysis technologies, NGS is very costly and data analysis can take weeks or even months to complete. A poor-quality library may yield poor sequence data or none at all. At a minimum, you risk wasting expensive or limited sample, and the cost of lost time on the sequencer can be even greater.
Different steps in NGS sample prep workflows require pipetting a wide range of volumes. In an automated NGS sample prep instrument, it is critical for the liquid handling pump to deliver the exact volumes required of each reagent at each step. The precision and accuracy of the pump are leading factors in ensuring high-quality sample and library prep.
A pump for automated NGS sample prep must deliver reliable, reproducible, and accurate results
Long-term research studies, crop and livestock strain improvement, and environmental studies require sequencing samples taken from different sources at different times. In clinical applications, diagnostic results must be consistent over time, among patients, and even across different medical centers. For all of these applications, consistent liquid handling pump performance is critical to help ensure that the NGS results are reliable and consistent every time, in every lab.
Consider these factors when choosing a liquid handling pump for your NGS application:
Testing each sample reliably and comparably test after test across labs is essential to measuring and monitoring results and trends.
2. Speed variability
Adjusting pump speed to match liquid viscosity, reagent administration and workflow times takes the guesswork out of testing/prep.
With samples at a premium, it’s essential to maintain their purity. A pump with disposable tips greatly minimizes the possibility of cross contamination and inaccurate results.
Ensuring that you are accurately dispensing or aspirating key reagents or target fragments is essential, because incorrect volumes can significantly compromise the results.
Pump flexibility enables the use of different NGS workflows and kits
Automated NGS sample prep instruments process a multitude of samples and kits, each with different reagents and protocols for specific sequencing applications. Each NGS workflow requires pipetting discrete samples and reagents at varying volumes, viscosities, and speeds. So many variables and requirements make selecting a pump for an NGS liquid handler especially challenging. Consider pump options that accommodate workflow flexibility, such as specific flow rates; various tip types, volume ranges and step sizes; and ports or valves that enable multiple fluid distribution channels.
NGS liquid handling pump performance and flexibility give a competitive edge
The NGS market is exploding, demand is increasing, and as a result new NGS applications are being introduced frequently. Even with this flood onto the market, the cost for NGS sample processing is still costly and time-consuming. Automating the NGS sample prep process is crucial – using the right liquid handling pump for this automation is imperative. Sloppy sample prep is unacceptable and avoidable.
Liquid handling pumps must live up to extremely high standards for precision, accuracy, consistency and flexibility. Automated NGS sample prep systems that accommodate various workflows and kits have a competitive edge in the market.
PacBio, a premier sequencing provider known for long-reads, demands excellence in their automated NGS liquid handling instrument. Their SMRT sequencing technology can deliver up to 99.999 % sequencing accuracy. With such high performance, you can be sure they don’t want to lose even a single thousandth of a percentage point because of a substandard liquid handling pump. PacBio chose the Cavro®Air Displacement Pipettor (ADP) for its performance, accuracy, and advanced features.
Read more about how PacBio is using the Cavro Air Displacement Pipettor (ADP) in their high-throughput sequencing workflow.