 Welcome to MOOC course on Introduction to Proteogenomics. In today's lecture, we will continue with Mr. Abhijeet Dixit, who will talk about the droplet digital PCR, which will be continuation of his previous lecture, where he started discussing about the basics of this technology. He will talk about the type of samples one can use as the input for the instrument. One can be used for multiplexing as well. Even up to 8-plex assays using single-flow cells have been reported. The principal requirement for multiplexing is the significant size difference between the ampli-cons. At least, there should be 30 base pair difference. So, let us now welcome Mr. Abhijeet to have an interactive session on digital PCR technology. Is everybody clear with the technology how it works? Anybody has any queries? Yeah, sure. The sample does not bind to the droplet, ma'am. Yes. So, these are droplets which are made where your oil is making a cover, right? And the sample and the reaction mix is inside those droplets. It is an amalgamation, missiles, in case you know what the missiles are. That is how it works. Any other queries? Right, ma'am. You would have to do extraction. You would have to do extraction, right? You can have your DNA or RNA from any source, does not matter, right, ma'am? You isolate your single cells by a flow cytometer or any other technology. So, what you can do is, even if you have a clump, even if you have say a pool of cells, right? You are wanting to look at a particular type of ampli-con. Now, in case of individual cells, then you will have to have a cell to extract a nucleic acid out of it. If you have a pool, how would the system differentiate between what cells it comes, the DNA or RNA coming from, right? Then you separate. So, you want to do a separation by this method? No, separation you will have to do by any other method. The PCR can happen here. The PCR and the quantification later part can happen in this. What you are talking of is also a different technology which we have, right? So, it is a workflow, ma'am, where you isolate your single cells by a flow cytometer and then go ahead with this, right, right, correct. What you are talking of, that technology is also there, which is a different technology altogether. I mean, we can have offline discussion. We have another technology which works for that. A single cell isolation and sequencing, yeah. At a time, you can have two flow, because there are two fluorophores, there are two lasers, you can have two, but there are publications where people have done up to eight plex acids with a single fluorophore. What you simply need to do is, keep the size of ampli-cons variable at least a 50 base pair difference between the two ampli-cons. So, what it would do is, yeah, now this cluster which you see is from one type of ampli-con, right. If another ampli-con is smaller or bigger than that, that would give a different layer here. You can quantify each of these clusters individually. For example, in 2D, you get it here, right. So, you will have another cluster altogether. So, you can have, I mean, there are a lot of publications where people have gone up to eight plex acids, sorry, yes ma'am? Or a multiplex? At least a 30 to 50 base pair difference. If you have to go more than 2 plex acids, right. It is not for single cell isolation. It is after you have a single cell isolation, because that amount of nucleic acid coming out of the single cell is going to be very minimalistic. So, the system is capable enough, sensitivity wise to capture even that miniscule amount of nucleic acid, a single cell, right, yes. It is not very difficult, ma'am. You can do a single cell isolation by flow cytometer very easily. A flow cytometer can do a single cell isolation very easily. And as I was mentioning ma'am, we have another system also, it is another technology altogether, where we can do a single cell right from isolation to this two sequences. That is the parallel technology different all together. It is an endpoint piece here. No, it is not. Yes. It is not a real time. No, what I meant was this is a real-time display how it comes. It is an endpoint piece here. Anything else? Any other queries? Yes. Yes. An ideal limit what we say is from a 60 base pair to a 350 base pair, right, right, right. It is actually not as I told. What is recommended is from 60 base pairs to 350 base pairs, like it is for real-time PCR as well. But we have got people who have amplified and specifically quantified up to 2 kbf fragments as well, very easily doable. What I am talking of are the ideal conditions which come from the literature which is published when it is being manufactured. What people have done, there are more than around 4000 publications now if I am not wrong with this technique. And people have done things which we even we could not imagine of. Yes. Yes. Absolutely. So, the prime requirement or the best amplification results which we will get with this does not depend on the quality of your nucleic acid. It depends on the specificity of your primers. If your primers are specific the data you will get is absolutely specific. The quality of nucleic acid would not hamper your data. Yes ma'am. No, it can be. You need to have you need to have a specific primer. Yes. Yes. You need to have specific primer for your amplification that you are looking for. And this is the fluorescence amplitude as each of your droplets is emitting a fluorescence. If there is a amplifiable DNA in the droplet it will give fluorescence, right. And when it emits a fluorescence it will be plotted here. If it does not emit it is here, right. But the system is optimized in such a way that your specific length of a amplicon if it emits a fluorescence it will emit a fluorescence in certain range. So, system takes care of those aspects as well. Is that what you just mentioned? Yes. Because as I told it is like a flow site or meter, the? 50. Yes. At least 30 or 50, 30 to 50 base per difference between two amplicons. Anything else? Technologically if I can answer some. Yes ma'am. The site before this ma'am? This? 1 cartridge. 1 cartridge. Right. Right. So, 96. Right. So, what? 8. 8 at a time for droplet generation. For droplet generation. So, this process this you are talking of, right. This takes 1 minute. Okay. So, you transfer your droplets in a PCR plate. You generate next? Yes. Yes. You are transferring these, right. You are generating your droplets which are here. In this top row to a PCR plate and then put a PCR. No, it does not happen. It does not happen. It has been well optimized for that. It has been well optimized for that. The droplets which are formed are very stable. You do not need that in this. You do not need that for this technology because each of your reaction is what is splitting into 20 dozen replicates. So, you do not need to have technical or biological replicates like you do for real time PCR. Yes. Samples of each of your reaction. DNA would not get divided. That your DNA is not getting fragmented. It is not getting fragmented. Right. First slide for this. This one. It was in the video. I would not be able to forward the video yet because I cannot see anything here. Yeah. Tell me a query, man. It is not one fragment, right. There are multiple fragments. Right. Right. Okay. Your DNA is getting, your DNA is getting fragmented, your DNA is not getting fragmented, it is getting partitioned. Correct. No, what is happening is your, you put your replicates or replicates to eliminate most of the times to eliminate your pipetting errors. If you go for a real time PCR, right, you should not have more than 0.5 CT value difference. Right. Right. So, here it does not matter because the data that you get is copies of your DNA per microliter of your sample. Right. It does not matter, sir. Not all droplets will anyway not have. That is okay. We have publications where people have got 0.0001% of the mutant as well. Right. Right. So, the data that comes out, the data that comes out comes after implying a Poisson's algorithm which takes care of your statistical significance. From the publication procedure to asking, there are more than 4 dozen publications already with this technology where this direct analysis has been done. Your term of asking is a SD, right, the standard deviation that you would get from each three replicates that you get. Correct. That is required because you might consider that your handling might be different, right, that we pipetting errors to take care of those things. Correct. So, but the drop, right, but the technology is made in such a way that that transfer of droplets, none of your DNA molecules are left behind. The technology makes sure that all of your reaction mix components are split up and are present in the droplets. None of it is left behind in the cartridge. The system is that rigid. It does not leave anything behind in the cartridge. Yes. None of the droplets what we make here, for example, in the first step, yeah, these droplets which are made, right, yes, yes. Everything is left behind in your cartridge. All of it is getting transferred. Nothing like is there back in the cartridge. The cartridges are not, you cannot reuse the cartridge. No, no. You cannot reuse the cartridge. It is a endpoint PCR, sir. So, that is what I mentioned, there was an entire slide on that, right, here, here. It quantitates it, the data that you get is it will give you how much ever DNA or RNA was present in your sample. It gives you copies of your amplified DNA in each sample, in copies per microliter. It is not a, you do not have to do any manual calculations like you do in real-time PCR. You have a CT value, then you do a FALF method, delta CT, delta, delta CT, do not have to do anything. The system quantitates and gives you the precise value, yes, ma'am, yeah. The minimum amount of DNA you mean, right. Again, we have got lots of data where people have worked on femtograms of DNA as well. In a single copy spike, you have a company called as Horizon, which gives you copy number controls, right. It gives you one copy, two copies, so you can spike your sample and check the copies. The system has been validated with that. The template can be of any size, but your Amplicon should not be very long, ideally should not be above a KB, right. So, in case you are using genomic DNA, right, we recommend doing a restriction enzyme digestion if you are using a genomic DNA, which you feel is going to be very long. Correct, correct. If it is a CDNA, then does not matter, but it is a genomic DNA, right, we recommend doing a restriction digestion, so that your template is available for the primers to bind and elongate. It will go in either of the droplets. It can be one droplet also, it can be 10 also, it can be 100 also, it can be 1000 also, does not matter, because it reads each of them individually. So, this thing, what you see here, yeah, even if you have this one droplet positive everything else is negative. You can be sure that that one droplet is because there is some DNA which is gone there, which is amplified and hence there is a fluorescence, yes, yes, yes. Above 5, generally above 10 lakh copies if it is there, then it starts because what will happen is all of it will be positive then, you will have no negative droplets. All of it will be positive. So, you might not get a very precise amount of DNA, that is about 10 lakh copies if you have that abundant amount of DNA in one sample. If you have per sample, per sample, per droplet because 20,000 into 5 becomes that number. So, per what happens is the system while it gives the data, it takes into account your forward scatter and side scatter like it does in a flow cytometer. So, even if there are up to 5 copies in one droplet, it quantifies them as 5 or 4 or 3 or 2 or 1, it does not give you as one copy. Maximum is 5 per droplet, that amount show more than 10 lakh copies in your sample. Anything else? Yes. So, in that case, yes, you would get multiple clusters here, you would not get a single cluster. Yes. If it is amplifying, is it binding somewhere else, right, the size of the amplitude variable, it cannot be of same size, right. So, it will give a different cluster here, you would not find such clean clusters, you will have another clear cluster which is which can be of a smaller or a bigger size. It is very sensitive for that, right. So, maybe in case you know one of your standard, even if you have one standard, for the first time if you are putting a new reaction, you have one sample where you know it is going to work for sure, right. You just put that for once and see which fluorescence amplitude does that come to for once and you know that value next time. So, if you are getting something variable, that means there is something non-specific also which is amplifying along with your specific thing. In conclusions, I hope today you have learned how do the PCR can be used for multiplexing, it has very high sensitivity and its specificity. DDPCR makes sure that the sample is not lost during the droplet formation, hence increases the efficiency of the process. In the next supplementary lecture, we will talk more about various proteomics applications. Thank you.