Don’t settle for an amplicon variant assay – build an amplicon sequencing assay

QIAseq 1step

There is a misconception out there that amplicon sequencing is beneficial only if you are interested in hotspots. Not true! Amplicon sequencing is beneficial regardless of the regions you are interested in, whether these are hotspots or entire coding sequences. There is a reason for this misconception, however. Let me elaborate.

The reason why amplicon-based sequencing has traditionally focused on hotspots is error rates – with every PCR cycle that’s used for amplicon-based enrichment, the chance of introducing an error increases, so does the chance of seeing and reporting this error in your data. At the end, the sequence that you see might be far from the sequence of the original DNA molecules you started with.

One way to reduce this error rate is to limit your analysis to specific spots within the region that you sequenced. How do you do this? Well, sequence the entire amplicon (say about 150 bases), and analyze and report only 1 base (the hotspot). In other words, design a ‘variant assay’. Statistically, the error rate has gone down tremendously. But, you just wasted 99.3% of your sequencing capacity (and $$) on bases you will never see because of error rate concerns! Now take a moment to think about your cost per base!

The alternative to the ‘variant assay’ is a ‘sequencing assay’ that reduces the error rates across all sequenced bases, not just hotspots, allowing you to see and interrogate all of them. Now, think about what this means. It means increased ability to discover new biomarkers since you are seeing more, and reduced price per sequenced base. In other words, you are using 100% of your sequencing capacity (and $$). How do you build a ‘sequencing assay’? You introduce Unique Molecular Indices (UMIs) in your workflow.

Amplicon sequencing panel figure 1

UMIs are tags that are incorporated into the original DNA molecules before any amplification takes place, thereby facilitating, bioinformatically, the identification of those original DNA molecules you start with. By analyzing UMI-containing reads, as opposed to naked ones, every sequenced base within an amplicon can be confidently interrogated due to an 8- to 20-fold reduction in error rates (see figure below).

Amplicon sequencing panel figure 2

Want to understand how UMIs work to reduce error rates? Click here!

Want to take advantage of UMIs to build your own customized ‘sequencing assay’? Click here to create your own panel!

Raed Samara

Raed Samara, PhD is a Global Product Manager for NGS technologies at QIAGEN, with a focus on pre-analytics and targeted enrichment. Prior to joining QIAGEN, he was a postdoctoral fellow at the National Cancer Institute conducting research in the field of cancer immunology with emphasis on identifying strategies to boost the efficacy of cancer vaccines. He received his Ph.D. degree from Georgetown University in tumor biology.

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