Why NGS Library Normalization is the Unsung Hero of Sequencing

When it comes to NGS library prep, there’s one step that can make or break your sequencing run: NGS library normalization. It’s the process that ensures every sample in your sequencing pool gets its fair share of reads—no more, no less. As Yann Jouvenot, Director of Product at N6 Tec, puts it:

“NGS library normalization is the step in the process where you ensure that all the libraries that are going to be sequenced are at equivalent levels. If you have some high abundance libraries and some very sparse ones, you’re going to have a huge imbalance… you’re really not going to have a good way to compare the data.”

With modern sequencers capable of churning out billions of reads, the stakes are higher than ever. If your libraries aren’t balanced, you’ll waste sequencing power and risk missing the data you actually need.

The Traditional Workflow: More Steps, More Stress

Let’s face it: traditional NGS library normalization is a pain. Labs spend hours purifying, quantifying (hello, qPCR machine and automated electrophoresis), and individually adjusting every sample. It’s a workflow that’s as varied as the scientists running it—and just as prone to error.

“Current methods involve individual purification and quantification for each library, which can be lab-dependent and time-consuming. These additional steps increase costs in terms of reagents, time, and analysis.”

Some labs even run pilot sequencing runs just to test if their normalization worked before committing to a full-scale run. Smart? Yes. Efficient? Not so much.

Meet iconPCR: A New Thermocycler for a New Era

Enter iconPCR, a next-generation DNA amplification platform for NGS that reimagines what a thermocycler can do. Instead of treating all wells the same, iconPCR features a PCR thermocycler with individually controlled wells, each with its own heating element and probe.

“We’ve completely deconstructed the concept of the thermocycler. Each well has its own thermal element and probe. We control the cycling of each well independently. That’s what enabled AutoNorm™.”

This breakthrough is what powers AutoNorm, iconPCR’s secret sauce for automated NGS library preparation.

AutoNorm: Automated NGS Library Preparation Made Simple

So, how does AutoNorm work? It’s as easy as setting your desired endpoint. iconPCR monitors DNA synthesis in real time using an intercalating agent (like SYBR Green). When a sample hits the threshold, that well stops cycling—no more, no less.

“AutoNorm normalizes libraries by monitoring DNA synthesis and stopping PCR for each well once a specified condition is met. You just take equivalent amounts from each well, pool them, perform a single purification, and load the balanced library onto a sequencer.”

The result? No more guessing, no more tedious quantification, and a single, streamlined workflow. Just pool, purify, and go.

The Goldilocks Principle: Why “Just Right” PCR Beats “Good Enough” Every Time

The pain point here was really the fact that, as mentioned earlier, NGS library normalization currently involves quite a few steps, is error-prone, and is costly in both time and consumables. But more than that, the development of iconPCR came from a desire to get better quality. By developing a PCR thermocycler with individually controlled wells that can stop normalization—stop cycling—at the optimum number of cycles for every sample, and do that automatically (thanks to AutoNorm), we prevent overcycling and undercycling in a way that manual workflows simply can’t match.

“If you ask any scientist in the genomics field, overcycling is the killer. It’s the killer of diversity. It’s the killer of sensitivity. It’s the killer of quality data. Nobody wants to sequence duplicates. Nobody wants to have to remove chimeras. Nobody wants to have to sort through artifacts that would otherwise be avoided.”

That’s the heart of the Goldilocks principle in PCR NGS: not too much, not too little—just the right amount for every well, every time. It’s the difference between “good enough” and “just right,” and it’s what makes iconPCR and AutoNorm a leap forward for anyone serious about NGS library prep—from Metagenomics and Spatial Transcriptomics to the trickiest FFPE workflows. Why settle for a workflow that’s merely working, when you can have one that’s optimized for quality, efficiency, and scientific confidence?

Real Results: From Metagenomics to FFPE, iconPCR Delivers

Early adopters are singing the praises of iconPCR and AutoNorm. Kerry Hair at Penn State University reports the instrument is saving her at least 50% of costs on library prep and that it’s been a revolution in her lab.

And for those working with challenging samples—think FFPE workflows, low-concentration RNA, or high-diversity metagenomics—iconPCR is a game changer.

“For degraded or low-concentration samples, you just set a desired endpoint yield, and the system automatically determines the necessary cycles for each sample to reach that threshold, removing the guesswork.”

The Future is Here: Automation and the Next Generation of NGS Library Prep

What’s next for PCR NGS workflows? Automation is the name of the game. Yann predicts:

“I truly believe iconPCR will become the norm for library preparation… It’s really designed to save people time, reagents, and most importantly, to preserve the quality of the data.”

As protocols adapt to this new generation of PCR systems, expect shorter, simpler, and more reliable NGS library prep—no matter your application, from Spatial Transcriptomics to Metagenomics.

Ready to experience the Goldilocks effect in your lab?

Discover how iconPCR and AutoNorm can transform your NGS library prep—making it easy, optimized, and streamlined. Reach out for a demo and see what “just right” really feels like.