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I typically don't need a mic, but I'll use one for today.
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So thanks everybody.
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I'm Luke Edelman.
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I'll try to take you guys through the pictures.
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I've never seen a PDF document format like that, but we'll go for it.
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The pictures are the more important part anyways for these sorts of things, so hopefully you guys can follow along.
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So we've developed a very new technology platform for single cell that works in fundamentally new ways compared with the technologies that you guys will be familiar with.
1:06
So if you look at the left here, we would basket most other single cell technologies on the market today as partition based technologies.
1:16
Meaning for the core step of labelling the nucleic acids of a single cell, you perform it by placing that cell into some sort of physical compartment or partition alongside the index that going to that you're going to use to label that cell.
1:29
And that partitioning process is where and how you perform the single cell indexing.
1:34
Now those technologies deserve quite a lot of credit for bringing single cell generally into the mainstream as a tool for molecular cellular biology.
1:41
But we think they're relatively constrained in terms of the next lapse of what single cell could look like in terms of scale, in terms of pricing regimes, things like that.
1:52
So on the right is our technology.
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Our technology is a solution phase technology, meaning it's partition free.
1:59
So I'll walk you through the key components of our technology on this slide on the right and then show you some particulars about the workflow performance data on the slides to come.
2:08
The entire workflow is done end to end just a normal laboratory plastic Ware like PCR tubes or strips or plates, depending upon the scale of the experiment that you're doing.
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And it's only normal liquid handling.
2:20
So you have a single sample and a single well carried all throughout the assay.
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There's no combinatorial split pool indexing or anything like that.
2:27
So in a liquid handling perspective, it feels basically like a bulk assay, even though it's actually a single cell technology.
2:34
There's two main technical components that I'll point out about our platform that basically are the key things that make it run.
2:41
There's a new type of indexing reagent, which is basically a bead technology and there's a new type of indexing reaction, which is a solution phase chemical biology technology that we've developed and brought together and they power our platform.
2:54
So on the left tube here, you see we have our indexing reagent beads.
3:00
These are sort of similar to the gel beads that you might be familiar with from other providers or other indexing bead technologies.
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Generally speaking, each bead has millions of copies of an identical index sequence, but the different beads have different index sequences.
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So the red bead will have millions of copies of the red index sequence, the blue bead millions of copies of the blue index sequence, etcetera.
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Each bead also has what we call cell binding moieties on their outer shell that make them physically sticky for the plasma membrane.
3:29
So they're directly cell binding beads rather than just indexing beads.
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As I'll say across the talk a little bit later on, there's two different versions of our cell binding chemistry.
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There's a universal version, which basically has generic affinity for anything with a plasma membrane.
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There's also a cell selected version which allows you to use antibodies or antibody panels as your cell binding moiety if you want to do in assay cell selection for single cell without needing to do a separate enrichment purification FACs MACs process.
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And that's useful for a variety of applications we're finding.
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So the very first step of the workflow, you add however many thousands of cells you have in a particular sample to a PCR tube or the well of a plate along with the corresponding number of these indexing reagent beads.
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Each cell and bead form 1 to 1 cell bead pairs driven by the cell binding moiety.
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So at the end of that process you have however many cells you have in your original sample, some fraction of which have been bound to a bead.
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We typically get capture rates on the order of 50% or so.
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That's tunable up or down by the number of beads, but typically it's roughly 50% capture at the midpoint.
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We then add a specially formulated lysis and indexing buffer and put the samples on a PCR machine for lysis and indexing.
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There's a couple different important things in that lysis and indexing buffer, but the most important of which is a high viscosity compound which retards the spatial diffusion of nucleic acids in solution during the process of lysis and indexing.
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And that generates what we call kinetic confinement and allows you to index thousands of cells in a normal PCR tube without partitions, just in a single step.
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So to bring it all together, you add your cells and beads to the tube.
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They pair directly driven by the cell binding moiety.
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You then put that on a PCR machine with your indexing buffer for lysis and indexing.
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There's a quick high temperature lysis step which lyses the cells, releases their nucleic acids and also releases the indexing oligos from each bead that was paired to a cell.
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So where you used to have a single cell paired to a single bead, you then have a cloud of nucleic acids composed of that cell’s nucleic acids and the indexing oligos from the bead that was paired to it.
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And you have that for each of the thousands of cell bead pairs that you have across your tube.
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We then drop the temperature to an indexing temperature which allows the indexing oligos to hybridise to their target nucleic acids.
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Our current chemistry is just a three prime gene expression chemistry.
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So that's a Poly A, Poly T hybridization to the mRNA tail.
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Now because that lysis and then indexing reaction takes place in this viscous buffer, each of these molecular clouds of a single cell and a single beads nucleic acids are kept in the very small tight confined volume and those confined volumes are where the single cell indexing reactions take place.
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Two last quick details about that process.
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Because these are small confined volumes, they create a high local concentration of nucleic acids which drives high efficiency indexing of the single cells to the single bead oligos, and it also prevents any sort of cross diffusion from one cell bead pair to another.
6:37
So that gives you high fidelity single cell, even though it's just in a single PCR tube and without any sort of partitions.
6:44
So if you see here, these are species mixing experiments.
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For those of you that are familiar with the technique for benchmarking and evaluating single cell technologies, there's four different samples here.
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Each has the same number of cells, the same number of beads and the same cell bead pairing reaction.
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But in each of these samples, we have a different level of viscosity, a different level of Kinel’ic confinement.
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Sorry about the text here.
7:07
Sorry about the text here guys.
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I didn't even know that pot was legal in Boston, but there we are anyways.
7:17
So you have on the left most sample here you have PBS for the lysis and indexing reaction.
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You have no viscosity at all.
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I'll point out that the numeric axes in each of these figures are identical.
7:26
So if you don't have any confinement, you basically get no signal at all.
7:29
You get is a little bit of noise.
7:31
And the reason for that is that the kinetic confinement is not only necessary for the high fidelity, it's also necessary for the high molecular efficiency of that hybridization reaction.
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So basically they you add your cells and the beads, then you do it without viscosity.
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The nucleic acids immediately distribute into a low concentration across your whole tube and you don't even have the kinetics to index any nucleic acids.
7:53
What if you go to increasing amounts of viscosity, increasing amounts of kinetic confinement, you see large numbers of cells, you see high sensitivity, you see high fidelity and doublet and noise profile rates that are generally in line with the partition based technologies.
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Even though this workflow is done just a normal PCR tubes just in solution base.
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This is an analytic demonstration that you don't need partitioning for single cell.
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You don't need partitioning or microfluidics or instrumentation to enforce high fidelity single cell indexing.
8:23
You can basically run it as a bulk assay with the right chemical biology.
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For those of you who are interested, we came out a couple of months ago with a bio archive preprint that walks in relatively deep detail through the tech, how it works, various applications and example data sets and so offline.
8:40
If you guys are interested in taking a deeper dive, you can feel free to do so.
8:45
This is what our kits look like.
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So we are new to market.
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We just launched commercially last quarter at ASHG in Denver.
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We came out with eight and 16 sample kits last quarter.
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We're coming out with 96 sample kits next quarter.
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96 sample kits will be targeted for either manual or automated workflow applications depending upon what you're looking for.
9:05
It's very easy workflow to automate since it's all basically just normal bulk liquid handling.
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It's compatible probably with basically any standard liquid handling robot that you have in your labs.
9:15
One thing that I'll point out is that regardless of kits size, the sample scalability is basically limitless.
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So say you get a 16-sample kit.
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You don't need to run all 16 samples at once.
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You don't need to burn the entire kit in the same single experiment.
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You can spread it across however many different individual experiments you may want.
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You can do all 16 at once.
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You can do 6 today, 10 tomorrow, 10 next week, whatever.
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And that's like a perhaps minor point, but actually we think that tracks more closely with how many people want to do cell biology rather than like forcing your experiment to fit the kit.
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In this case, you have pretty significant flexibility to architect the biology how you want and then the kit can fit that.
10:01
It's just a couple of boxes.
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There's no accessory instrumentation or anything.
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All you need is the kit and a PCR machine like a magnet to do clean ups and stuff like that.
10:09
So it's pretty straightforward.
10:12
The workflow is pretty simple and pretty fast.
10:15
It's 8 hours to go from your input cells to sample multiplexed and sequencer loadable libraries.
10:21
There's a variety of pause points across the workflow.
10:24
One pause point that I'll point out in particularly is quite early on.
10:29
So after you pair your cells to those indexing beads and put them in that viscous buffer, but before you do lysis or indexing or any of the subsequent molecular biology or library prep, you can actually just flash freeze the paired cells on dry ice or put them in -80 we've added a cryopreservative molecule in that viscous buffer that keeps the cells stable, keeps the beads stable, keeps them bound to each other.
10:55
And you can use that to separate your sample acquisition from the 80% of the actual single cell library prep reaction.
11:04
A lot of people use that just in a simple manner of you get your cells at some point on one day.
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You don't generally want to do like a big chunk of molecular biology after that, it would be a long day for people.
11:17
So a lot of people tend to use this first fixation free pause point just to do the first like 90 minutes of the workflow.
11:23
Get your cells, prep your cells, pair them to the beads, put them in the freezer and then go home for the day.
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Then you can do the other six hours or so of the library prep thereafter whenever you want.
11:32
And you can freeze them that way for up to several months in fact, but it can also be as short as several hours.
11:39
So quickly flashing through some data, it works on a pretty big variety of cell and specimen types, cultured cells, solid tissues.
11:48
It works very well on blood or PBMCs.
11:51
Like any single cell technology, there's a little bit of optimization that you generally want to do to ensure you get the right capture rate and the cells and samples are of the high quality.
11:59
But it works for a pretty broad array of cell types.
12:04
This is an example of doing relatively large number of samples just by hand.
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We had one of our R&D scientists process 496 well plates in a single day from cells to sequencer libraries.
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This was a long day.
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It was like 10 or 12 hours.
12:18
And this dude made it clear that he wasn't keen on doing that every day.
12:21
But nonetheless, if you wanted to do 496 well plates in other technologies, it would take a relatively long period of time and be a somewhat unpleasant time.
12:30
And for those that are considering much larger experiments, it's very amenable to automation and the scales in terms of samples would be even larger.
12:37
Again, I might quickly skip over some of the data.
12:41
We have a variety of early customers that have used it across a variety of some specimen types.
12:45
What I'll highlight in specific is this small life science company on the left that want to do 10s to hundreds of thousands of samples of PBMCs or whole blood.
12:55
They did pretty extensive benchmarking with our technology before they ended up committing to us, including looking at known cell pipes and making sure the right transcriptional markers are there, cross comparing our tech against normal flow, looking at various measures of reproducibility and reliability.
13:12
And they also compared us in a biologic sense against another leading on market single cell platform.
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And even though our technology works in a very different way, even though the analytic performance measures in terms of like sensitivity or cell number are different, you actually get more or less the same underlying biology.
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So you get the same overall cell type clusters.
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You get the same clustering patterns, you get the same pathway enrichment if you look at different cell types and you're looking at intracellular transcriptional programming.
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So even though our technology works in very different biophysical ways compared with the partitioning based technologies, actually the biology, the insights, the actual discovery that you receive at the end of the day is more or less identical.
14:03
This is just another early application from a customer I'll skip over that.
14:07
The last quick data slide.
14:09
I mentioned earlier that we have the cell binding moieties on the outer shell of each bead.
14:14
Those in our core standard chemistry are universal, but we can also make that cell binding interface cell specific by dropping in one or more antibodies against cell types of interest.
14:24
In this example, we've simply done PBMCs either with a pan PBMC antibody, which as you see basically grabs all cells in the PBMC compartment or with a T cell or AB cell specific antibody respectively.
14:35
And you see, you know, not the same purity that we get with like a good FACs experiment or something like this, but you see pretty good like fold enrichment for T cells or B cells specifically.
14:45
And this is something that could be useful for a variety of applications.
14:47
This doesn't add any complexity to the workflow.
14:49
In fact, it's slightly for specific reasons.
14:51
It slightly reduces the time of the workflow.
14:54
So it's still like a 7/7 hour day basically, but it allows you to bring the cell targeting into the assay without any additional liquid handling.
15:03
This could be useful if you want to target specific cell types without needing to FACs or MACs.
15:07
It's also useful if you want to target specific cell types that tend to disappear in other single cell technologies.
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Would be a good example.
15:14
We have a relatively large number of people that want to do large scale neuronal screening, either with cultured cells, organoids, 3D cell cultures or primary cells.
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Microglia of different sorts is a very common cell type of interest for therapeutic discovery, but they are fickle cells.
15:30
They tend to disappear from your data after you do single cell.
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We're working with a couple of partners on a microglia specific capture bead, so you have the right biology, the right insights, so you can do that at scale.
15:43
So I will pause there as the last slide here.
15:47
Hopefully this has been interesting to you guys to learn about a new approach to single cell that's pretty scalable, pretty flexible.
15:55
And if you want to hear more about it's also pretty affordable.
15:57
So you should stop by our booth if you want to learn more.
16:00
Thanks so much.