Library normalization is crucial for optimizing clustering and data quality – but what is it? Pedro Echave, Global Leader of NGS Product Experts at PerkinElmer, explains, “Normalization refers to the process by which libraries of different concentrations are diluted to the same final – ‘normalized’ – concentration before pooling and loading into the sequencer. This step is recommended to obtain best data quality from a run, so most next-generation sequencing (NGS) users do it. To normalize, you must quantify your libraries, do some calculations, and adjust the concentration of each library by pipetting different volumes of buffer (usually manually).”
Though normalization is crucial, the potentially convoluted process places time and cost constraints on labs – particularly those feeling the strain of workforce shortages. That’s where PerkinElmer’s new, patent-pending, bead-based normalization technique comes in. “Bead-based normalization uses a completely different approach to yield the best data quality,” says Echave. “Here, DNA-binding beads are added to all the libraries. A fixed amount of DNA will bind to those beads, which means that, after elution, the libraries will have approximately the same concentration. Therefore, by using bead-based normalization, you eliminate the need to quantify and dilute your samples before pooling.”
How, then, can bead-based normalization be applied to NGS? “In theory, bead-based normalization can be used at different points during library preparation workflow, but it is mostly applied at the end – after libraries are finished,” says Echave. “The user adds the beads, elutes them, and then pools the samples in a new tube by adding equal volumes of each.”
Choosing bead-based normalization for NGS has many benefits for lab medicine professionals – key among them reducing turnaround times and saving costs. “Bead-based normalization is faster and less labor-intensive than traditional normalization methods,” says Echave. “Additionally, traditional normalization requires reagents and consumables (such as Qubit reagent, tubes, tips, and microwell plates) that are not needed when using beads.”
The method can also help with multiplexing and scale laboratory capacity, which could alleviate shortages in the laboratory workforce. “The main advantages of normalization beads are the savings in reagents and consumables – but, by also reducing the time required to load samples into the sequencer, normalization beads allow laboratories to process more samples,” echoes Echave. “With normalization, laboratories can save up to three hours per 96 samples, which increases processing capacity and throughput. If the lab can process a higher number of samples, this will yield a bigger time-saving benefit for them and, in turn, decrease the cost per sample.”
A particular application example that comes to Echave’s mind is using PerkinElmer’s NEXTFLEX® Variant-Seq™ SARS-CoV-2 Kit with normalization beads. “During the pandemic, laboratories were flooded with thousands of SARS-CoV-2 PCR-positive samples to sequence so that they could track the emergence of viral mutations in real time. There was a lot of pressure to deliver results as quickly as possible; however, the yields of libraries from SARS-CoV-2 PCR positive samples are highly variable – a direct consequence of the range of viral loads that appear. That’s where normalization beads help. The beads in our NEXTFLEX Variant-Seq SARS-CoV-2 kit v2 successfully normalize libraries with Ct≤32, which saves up to three hours of processing time for every 96 samples – effectively reducing turnaround times for labs at a time when it is sorely needed.”
With the advantages of normalization beads clear, how else could labs improve their NGS workflows? Echave suggests, “To be more cost effective and yield rapid, reliable results, labs will need sensitive kits and optimized and automated NGS workflows that are adapted to their throughput and applications. When starting their journey to more effective workflows, pathologists should know that PerkinElmer is happy to support its customers with optimized solutions and application expertise.”
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