Capturing Sequencing Data from Rare Transcripts and Low-Copy Variants

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Andrea Corona: Hi, everyone, and welcome to Biocompare’s Tech Insights Podcast where we speak to scientific experts about new tools and technology that can help advance your research. I'm Andrea Corona, Associate Editor here at Biocompare. Today's guest is Liz Carpenter, Senior Support Scientist at Roche Sequencing, and she's going to be talking to us about the KAPA HiFi DNA polymerase for research use only, not for use and diagnostic procedures. Thank you for joining us today, Elizabeth.

Elizabeth Carpenter: Thank you for having me.

Andrea Corona: Of course. Well, I'll go ahead and get started with my first question. Don't all DNA polymerases have a proofreading function? What makes KAPA HiFi special?

Elizabeth Carpenter: So no. Not all DNA polymerases have a proofreading function. Enzymes generally fall into one of two families. And in short, one family has a proofreading function and the other one does not.

Typically the family of polymerases with a proofreading function are less efficient and not quite as robust, but they do have a lower error rate and developing KAPA HiFi, we were able to generate a polymerase that has a lower error rate with a high efficiency and can also amplify longer fragments.

Andrea Corona: So KAPA HiFi DNA polymerase was created by directed evolution technology. What does that mean, and why does it matter?

Elizabeth Carpenter: Directed evolution is like natural selection in a test tube. We use a combination of random mutations and artificial selection pressures to evolve the enzymes with improved properties. This method allows us to engineer improvements to an enzyme irrespective of what the enzymes were actually designed for. By imposing these background pressures, it enables us to select polymerases that are generally very good at PCR.

Andrea Corona: So what does it mean for a DNA polymerase to be processive? What attributes go with processivity?

Elizabeth Carpenter: Processivity is the ability for an enzyme to continuously incorporate nucleotides without dissociating from the DNA fragments. When an enzyme has high processivity, you're able to amplify much longer targets.

Andrea Corona: My sequencer does approximately 150 base rates. Why would I need a process of DNA polymerase that can amplify up to 1100 bases? What can that do for me, and what applications does that support?

Elizabeth Carpenter: So if you're sequencing shorter fragments, you won't actually need a polymerase that can amplify long regions. With KAPA HiFi, it's the other properties such as high fidelity and high efficiency that would be of more interest to you. But as sequencing technologies progress and longer read sequencing is more prevalent for applications such as whole genome sequencing or de novo sequencing, you would also be able to use KAPA HiFi because of its high processivity.

Andrea Corona: Why do speed, range, and the ability to tolerate inhibitors tend to go together in a typical polymerase? And why does this generally mean that, as a result, it gives up accuracy?

Elizabeth Carpenter: So in all amplifications, the end goal is to replicate a specific region or target. With current workdays being a set number of hours and various sample types being studied, it's essential to have enzymes that can overcome those obstacles. And depending on the circumstances, compensating for one may mean losing on another. And most often, as you said, it's fidelity. Typically because in order for an enzyme to work faster, and be somewhat forgiving, when it comes to inhibitors, the enzyme will most likely be less stringent when it's incorporating the nucleotides.

Andrea Corona: So KAPA HiFi DNA polymerase boasts both high fidelity and high processivity. How does that lead to high sensitivity? In other words, how can that help me to detect rare molecules in heterogenous samples? How can it help me to capture sequencing data from rare transcripts and low copy variance?

Elizabeth Carpenter: When an enzyme can accurately incorporate nucleotides on longer fragments, this increases the potential to detect rare variants as it allows for deeper sequencing with a lower error rate. And it can cover a larger region and this, in turn, lends to high sensitivity.

Andrea Corona: So my last question for you is how does KAPA HiFi DNA polymerase help ensure an NGS library with uniform coverage?

Elizabeth Carpenter: This also ties into the previous question a little bit. KAPA HiFi’s ability to accurately incorporate nucleotides, especially over difficult areas to amplify, such as high GC regions helps to decrease bias. And by decreasing the bias, you can have better coverage across all areas of the region you were wanting to amplify.

Andrea Corona: Well, that was all the questions I had. So thank you so much, Liz, for sharing your time and knowledge with us.

Elizabeth Carpenter: Thank you. It was my pleasure.

Andrea Corona: And if you want to know more, please read the related article titled KAPA HiFi DNA polymerase Combines High Fidelity and High Processivity and thank you so much for listening. For more information on products, technologies, and the latest scientific advancements please visit biocompare.com and have a great day.