In this webinar, we explored how transposase-based library technologies are well-suited for a number of challenging high-throughput workflows, and how innovations such as purePlex, ExpressPlex and Tagify are accelerating critical sequencing-based applications.

Introduction

seqWell was created to revolutionize NGS library prep to unlock the full potential of today’s DNA sequencing instruments, and Tn5 transposase-based solutions are critical to optimizing that potential. This enzyme has become an integral component of NGS library prep. Its unique capabilities have unlocked a plethora of recent advancements now at the forefront of biomedical research and development.

This article is the first in our series that highlights seqWell’s multimedia content offerings. The following is a transcript of our recent webinar: Enabling Sequencing Applications with Improved Transposase-Based Solutions.1 Jessica Smith, PhD, Director of Bioinformatics at seqWell, moderated the webinar. She was joined by Joe Mellor, PhD, seqWell’s Co-Founder and Chief Scientific Officer, and Curtis Knox, seqWell’s Director of Product Management.

Learning Objectives

Jessica Smith: Curtis, what can our viewers expect to learn today?

Curtis Knox: Sure. By the end of this discussion, we hope that you will be able to, first, understand how tagmentation can work across a wide variety of NGS applications. Secondly, we want you to understand how transposase-based NGS library prep methods can result in significant workflow improvements. And then lastly, we think that you’ll be able to understand how some custom Tn5-loaded reagents can create opportunities for new innovations in your laboratory and be able to do all of this without compromising your data.

Background and Biology of Tn5 Transposase

Jessica Smith: Joe, do you want to give us a little background on Tn5 in NGS?

Joe Mellor: I think it’s helpful for us to start with an overview really, of where the sequencing arena is today. The growth of sequencers has become so prolific. We are now using instruments that are orders of magnitude higher throughput and larger than they were even a few years ago.2 And because of that, sequencing has really become a universal tool in life science research, not just for sequencing genomes in other samples, but really for a variety of types of measurement of biological assays.

It’s important to point out that the cost and time associated with getting samples to those sequencers, which we call library prep, has really lagged behind and has not advanced and kept up with the tremendous improvements in sequencer throughput. seqWell’s mission is to focus on that bottleneck: How to create the tools that will enable researchers and users of sequencers to get to sequencers more efficiently. In many cases, that means we enable higher multiplexing of more samples and simpler workflows to allow samples to be processed more quickly and efficiently.

Jessica Smith: Thank you so much for that background. I think it really does sort of set the stage for this conversation. Do you want to get into a little more detail about the biology of Tn5?

Joe Mellor: Tn5 is a centerpiece of most of our products here at seqWell because it’s an extremely versatile and a simple tool for helping scale a number of NGS workflows. Before we get into those products though, I think it’s helpful for us to maybe take a little bit of a step back and talk about the history of Tn5.

Tn5 has been around for a few decades now, has been highly characterized, has a number of really interesting properties that owe to its natural capability of being part of a transposon. Transposons are mobile genetic elements in genomes and this enzyme was evolved, essentially, with the capability of allowing that DNA to transfer around inside a genome.

Jessica Smith: Now, can you go into a little more depth about how Tn5 is useful for NGS applications?

Joe Mellor: There’s a few interesting properties about Tn5 that make it a versatile enzyme for NSG. I think probably the most important is that it’s capable of binding and inserting DNA nearly randomly in any target sequence, which allows it to be substituted for protocols that need a shotgun fragmentation-like function.

Another interesting property of the enzyme is that it can be functionalized, loaded with different types of payloads, which we have turned into the ability to put different sequencing type functions into it, such as sequencing adapters.

Finally, I think the other piece that we’ll talk about today is that the enzyme has a property that allows it to be exploited as a normalization agent because its function is to do its job and do it one time only, and therefore controlling the amount of enzyme can control the number of reactions that occur in a typical reaction.

Leveraging Transposase-Based Solutions for Optimized NGS

Jessica Smith: Curtis, tell us a little bit more about the various ways that we can use this technology in different NGS library prep applications.

Curtis Knox: Sure. Joe talked about the biology of the enzyme itself, and what I want to talk about a little bit is how we’ve used that to create NGS library prep methods and products that really help the users when it comes to getting the most efficiency out of what they’re trying to do.

What we’ve done is create a series of methods and products that allow for what we refer to as true multiplexing, much higher levels of samples that can be multiplexed together for their sequencing. And we do this through a method in which we’re utilizing asymmetric tagging of the libraries, adding the i7 section and the i5 section in sequential fashion. And that allows really nice control, allows normalization that Joe alluded to, and has high efficiency for converting the DNA into actual sequencing libraries.

As an example, when we think about traditional ligation or other types of methods, you’re typically looking at five and a half, six hours or more to do a plate of 96 samples. With our methods using Tn5 transposase, we’ve gotten that down to, right now, three and a half or less hours to do that full plate of samples. And, at the same time, creating some of those extra bonus features like the normalization that we will talk about. And these have a wide range of applications, everything from whole genome sequencing, low pass genotyping, microbial and metagenomic sequencing, and even single cell RNA-seq from there.

Jessica Smith: You mentioned normalization as a bonus, but it actually seems pretty central to a lot of the products that we sell. Can you go into a little more depth about this unique capability and give us maybe an example of one of our products that leverages normalization?

Curtis Knox: The first thing I’d like to maybe use as that example is our purePlex DNA Library Prep Kit that we just released earlier in 2022. And this is one where we’ve taken those features and actually improved the workflow down to as little as two and a half hours when it comes to that. And we’ve also incorporated unique dual indices into that kit.3 That’s something that we’ve heard from users that they’re looking for: extra control of their samples and data quality and everything else from there. The incorporation of the UDIs has been very important as well. That is a product that currently we have with 192 different UDIs. We plan to expand that up to about 384 in the near future, as well.

Jessica Smith: You mentioned briefly that UDIs have something to do with data quality. Do you want to clarify that a little bit?

Curtis Knox: When you think about making sure that the sequences that you get are definitively coming from each of your samples that you’re interested in, because those samples all get pooled together on the sequencer, it’s very important that you be able to assign those reads exactly back to the sample of interest. Utilizing unique dual indices allows you to make sure that you’re fully separating that data out and have the highest confidence.

Jessica Smith: We’ve started talking a little bit about how our multiplexing capabilities can lead to improved performance. Joe, can you discuss how normalization works in practice?

Joe Mellor: The idea of normalization is that in many sequencing workflows, the user, the researcher, has their sample and maybe it’s present in different amounts. It’s a difficult problem and labs spend a lot of time with this.

The way that our chemistry works is that it facilitates a sequential tagging process where the first tagging event is used as a way of specifying the number of molecules that can be generated. And then we pool that material together, or in the case of purePlex, we have a different method for accomplishing this, to then turn those first tagging instances into a normalized library where all of those samples are represented with the same number of reads. The point is, with our chemistries, this process is highly simplified, and we’ve removed, for many people, this need to do a separate normalization step.

Transposase-Based Solutions in Action

Jessica Smith: I can imagine a lot of different clinical applications where this would be useful. Please share an example of how our multiplex workflows are used in the real world.

Joe Mellor: I think there’s no better example in the last couple of years than labs that have had to scale up, for example, the ability to sequence COVID genomes.4 I think all of us woke up to a need, really overnight, starting two years ago, where we wanted to be able to track and characterize different variants of SARS-CoV-2 genome that was emerging.

An example of this type of work was a collaboration we did with a group at TGen in Arizona where essentially, overnight, we helped them stand up a COVID sequencing effort where they could take their COVID-positive specimens and reflex them into a sequencing assay and track those variants in real time to better understand what was going on with the spread of the virus and new variants.5 I think this is a great example where a lab was able to go from doing zero of this type of sample to thousands per week in a matter of a few weeks.

Jessica Smith: That’s a really great example of the power of this kind of technology. Now, let’s look ahead at some new products we have. Curtis, do you want to tell us about what’s coming down the pipeline?

Curtis Knox: I think first maybe, Joe, if you’d like to touch on some of the rapid single cell RNA-seq applications, and then we can follow that up with some of the really exciting stuff that we’ve got coming down the pike here.

Joe Mellor: Yeah, absolutely. As we’ve taken the plexWell and purePlex technologies as the basis and foundation for other types of assays that we can build, we look for applications where this need to multiplex or essentially process large numbers of samples is important. And one really good example of this is in single cell sequencing, especially single cell RNA-seq.

And again, we collaborated with a researcher who specializes in this type of assay, was actually the inventor of a method called SMART-seq2 about 10 years ago, Simone Picelli.6 And we created a kit that allows for this integrated processing of single cells through to a sequencer ready RNA-seq library in essentially a single day. I think the story of this product is really that we were able to take the plexWell technology and combine it with a high-performance chemistry to essentially take RNA from cells and turn it into the DNA that becomes the input to our plexWell kit which has, again, this really important property of normalizing for the differences that might exist between those samples.

If you look at the performance of this product, this is where it really shines. Again, in a conventional sort of setting, the researcher would have this problem of having some cells generate more data than other cells, and they would have dropouts, for example. With plexWell, this problem is significantly improved because you get, again, greater performance across a greater number of samples and essentially more sequencing data from more cells in the same assay.

And I guess the final piece of this is that we do this at really no ultimate cost to the actual sequencing performance itself. It’s a workflow improvement. It certainly enables the multiplexing side of this. But if it produced worse data, this would not obviously be a great solution, we know, for scientists. And so, we’ve benchmarked this and we see that again, we can have this really nice normalization without any real cost in terms of the metrics associated with detecting RNA from cells.

Emerging Innovations

Jessica Smith: Between the COVID and single cell work that you just described, it seems like a lot of solutions are being generated by our company. I’d love to hear about what sorts of things we’re going to help other researchers with.

Curtis Knox: There’s some really exciting stuff coming down the pike, including what we were referring to now as ExpressPlex, which is something that we have in early access at the moment and we’ll be rolling out to users in the near future. And this is, by far, the fastest overall NGS library prep method out there to be capable of this true multiplexing and high levels of multiplex that we’ve talked about.

And in this particular method, you literally are mixing your DNA, a tagging reagent, and a master mix. You’re putting those three things together, putting on the thermocycler for an hour, and then you’re pooling and doing one cleanup.

In 90 minutes flat, you have 96 samples ready to go, all barcoded and ready to pool and put on your thermocycler. We can continue that barcoding, multiplexing aspect upwards. We anticipate 768 barcodes in the near future, moving that upwards to 1,536 and beyond, as necessary, from the users as we find that out. We’re really excited to bring this to customers in the very near future.

Jessica Smith: Going back a little bit more to the transposase itself, are there any ways that we’re leveraging that technology to do multiple different applications?

Curtis Knox: That’s a great segue into talking about something that we’re referring to as Tagify, which is a Tn5 transposase-based toolbox, for lack of a better phrase, in which we are going to help users and researchers out there utilize the power of transposase methods and enable different types of innovations utilizing the enzyme along with some different tags to actually achieve new things. And that can be something like CRISPR verifications. It can be looking at variations where we’re enabling ligation methods.

We have unique molecular identifier methods and versions of this in which we’re loading on sequences that’ll allow customers and users to really be able to home in exactly down to almost a molecular level and look at the sequences that are coming through. It’s a super exciting new thing that we’re pushing into to help enable users to really create even more innovation in their laboratory.

Jessica Smith: It’s wonderful that we have this really flexible transposase-based offering. I wonder, Joe, if you have a little bit more to say about the application areas and the technical details about what we can do to help our customers?

Joe Mellor: As Curtis mentioned, we’re working in this Tagify arena. It’s about creating tools that, again, our users and those researchers can take and plug into assays that they’re trying to build.

We have some great products that help in multiplexing, but again, some of these are also just basic tools that can help build the next generation of multiplexing tools. And one great example of this is, again, the CRISPR QC methods, which benefit often from being able to put a molecular counter into the reagent that’s being used to generate a library.

The tried-and-true way that this field has used for many years is to do a fragmentation and ligation-based assay. This is a great example where transposase can come in and take what was previously several distinct steps in a workflow and replace it with a single step that has equally high performance and simpler workflow and more predictable performance.

For this field to mature, there’s going to need to be more tools. We’re really excited to have the opportunity to be involved in building those tools.

Jessica Smith: You mentioned more tools. I should be clear that most of the work that we’ve talked about so far has been on Illumina platforms. Everyone knows Illumina, but we also know that there are more tools coming into the sequencer space, too. Do we have any plans to integrate our library prep with those other sequencers?

Curtis Knox: Absolutely.

Joe Mellor: Yeah, definitely. This is a really exciting area. The history of NGS till this point has really been the story of one very dominant sequencing platform, which has really built this market. But we now see, and I think it’s exciting, the emergence of other sequencing platforms with, again, slightly different chemistry, slightly different abilities that are going to give researchers and NGS users more choice in what types of platforms but also really specifically tuned perhaps to certain assays that they want to run.

We’ve been very excited to start to work with some of these newer platforms. For example, we collaborated with Shawn Levy and his team at Element Bioscience to validate our plexWell technology on their new AVITI sequencer.7 And we’re very excited to see this technology. We see that it certainly has a couple of benefits over existing sequencing platforms in terms of its flexibility and cost and some metrics that are important such as duplication rates and so on.

As these technologies progress and other sequencers are made available, we’re certainly, I think, going to be in a good position to use some of the same library prep technologies that we’ve built into our kits. And we think multiplexing will be an important part of all these platforms. We know that, again, users and scientists simply want to be able to run more samples and do so more reliably. And so we’re very excited about that.

Closing Remarks on Transposase-Based Solutions

Jessica Smith: I helped out a little bit with some of the Element work, and I have to say the metrics that came off were really impressive. It was really interesting to be working on.

All right guys, I’m going to let you both say at least one more thing about transposase and library prep if you’d like. Joe, I’ll let you go first.

Joe Mellor: As you’ve probably learned today, Tn5 transposase is a real centerpiece for our work in building better NGS workflows. And we think it has some intrinsic benefits that, for example, enable more scalable performance. And again, inherently address some of the core issues that users face every day, like how to process more samples, save time and money and effort associated with things like normalization.

And what you’re going to see from seqWell as time goes on is, again, we’re going to be taking some of the same approaches we’ve taken to date in terms of taking workflows that would otherwise be more complicated or take more time for a user and build workflows that essentially remove steps or simplify steps, for example, with new formulations of these enzymes, combining them with other enzymes as we learned about today with ExpressPlex. Again, very exciting product where we’re able to essentially do many things in one reaction mixture. I think this is going to be a familiar theme in how we engineer workflows to do the next generation of assays.

And certainly as far as the Tn5 enzyme itself, which is really the star of our show, I think we’re very excited on all of the improvements that we see that lie ahead in terms of what we can do with that enzyme.

Curtis Knox: And I would just add to that, hopefully people have seen and can maybe understand that we’re still scratching the surface. There’s still a lot of improvements, a lot of advancements that we’re continuing to make using Tn5 transposase as the base module for that.

It’s not just about what we produce and put it in a box, it’s about helping our researchers and our collaborators to make new innovations and to do great science.

We’d encourage anybody who’s interested in learning more about this or wants to talk about a project and say, “What if?” to reach out to us. We’d love to have that conversation with you and see if we can’t help.

References

  1. https://seqwell.com/video/enabling-sequencing-applications-with-improved-transposase-based-solutions
  2. https://seqwell.com/sanger-method-the-mp3-player-of-sequencing/
  3. https://seqwell.com/when-unique-dual-indexes-matter/
  4. https://seqwell.com/how-whole-genome-sequencing-changed-our-approach-to-covid-19-pandemic/
  5. https://www.tgen.org/news/2021/july/15/tgen-looks-beyond-covid-19-as-it-advances-pathogen-sequencing/
  6. https://seqwell.com/video/resolution-at-scale-plexwell-rapid-single-cell-for-scrna-sequencing/
  7. https://seqwell.com/seqwell-and-element-biosciences-partner-to-demonstrate-sequencing-performance-of-multiplexed-plexwell-technology-with-the-aviti-system/