PanelPlex demo (multiplex pcr design software)

This is the login to our DNA Software application
portal. It’s easy to register if you haven’t registered
already. You come into the portal and you can see our
three products that we have: qPCR CopyCount, which is for analysis of PCR to get absolute
quantification; ThermoBLAST, which is a stand-alone product that you can use for checking primers
that you may already have at your organization; and PanelPlex, which is what I’m talking about
today. Let’s go ahead and design some solutions with
PanelPlex by clicking on the blue button.Let’s go ahead and now run PanelPlex. PanelPlex has four different steps. First we need to choose our panels, our playlists,
the Inclusivity, Exclusivity, and Background. Second, we to choose this thing called the
“keystone”, which I’ll explain in a moment. Third, is entering the details of the target,
either do design to the whole target or a region of the target? Forth, we’ll give the hybridization condition
and any probes modifications.Here are the basic steps for doing the design in PanelPlex. The first is that we need to choose our panels. I’ve already set up Inclusivity and Exclusivity
lists for the Zika virus, so they’re already here. We have a separate webinar that we’ve given
on ThermoBLAST. This is the same interface here that we have
in ThermoBLAST for creating playlists and for accessing them. For today’s talk, I’m not going to talk about
how to create the playlists today, it’s very simple. Here’s the Inclusivity playlist for Zika virus,
for example. It has all 168 Zika virus variants, and here
they are. We’re going to choose that to be our Inclusivity
list for today. Just hit the link for ‘Select as Inclusivity
Playlist’. Now, we have to choose the Exclusivity playlist. I’ve already prepared this Exclusivity playlist
for Zika, so let’s go ahead and use that one. This Zika exclusivity playlist has 5,443 near
neighbors of other Flaviviruses and fever causing viruses that are somewhat related
to Zika virus, that we want to make sure don’t cause false positives. For example, we can see some examples of Dengue
fever virus, Chikungunya, which is actually not so related, it’s a Togavirus, but it’s
still a fever causing virus, and several other viruses that you can see here in this list
that are related to Zika virus, that we would not want to cause a false positive. That has been pre-done, so I just choose that
as the Exclusivity playlist. We’ve now chosen our Inclusivity and Exclusivity
playlists. The last step is to choose the background
playlist. In this case, I want to make sure that the
Zika diagnostic that I design does not bind to the human genome. So the human genome, I choose that as my background
sequence. I just did a little search here of all our
playlists, I just listed every playlist that involved the word “human” and here they
all are. The first on the list is human genome, so
I selected that. In summary, I’ve selected my three different
playlists, and those are going to be used by the software to determine the coverage
and the background. All 4 problems of multiplex design that we
talked about, are going be addressed by the software, PanelPlex. Next is this issue of the keystone. I haven’t talked to you about what a keystone
is, but here it is. There’s a definition here right at the top
of this page. The keystone sequence is one of the sequences
from the Inclusivity playlist that you want to detect with primers that have no mismatches. Typically, you would choose a reference genome
that is biologically or historically considered to be the most important sequence in your
list. Now, we have three different ways that you
can choose the keystone sequence, which this is the sequence that is going to be sort of
the center of the design and variants thereof will go around that design.Even if you choose
a bad keystone, the software is smart, it will try other keystones as well and compare
them to yours. If you hit the ‘Suggest Keystone’, it will
try several different keystones, including yours, to see which one works best. In this case here, I have a starting keystone. That was what I had cued myself to remember. This is just showing you the complete list
of Inclusivity sequences, now I put the keystone accession in the search box. Next, we go to the “Target Details” page. We need to give this job a name. I’m going to call it the ‘Zika demo two’. Next, we choose what type of detection we
want to use. If this was an NGS application, we would choose
no probes because you just want to amplify the target. If we want to detect them with a fluorescent
probe, like a TaqMan probe, we choose probe here. The next option is do you want to detect the
whole target, in which case the software will check the entire Zika virus, which is over
10,000 nucleotides in length, and it will try to find the best places for design. Or we can choose a design range. We allow design ranges up 40,000 nucleotides. In this case, the Zika virus is 10,000 nucleotides. We could say, “Oh, please direct our design
to where we know where there’s a gene,” maybe it goes from position 1,500 to 2,500. Thus, PanelPlex would limit the design to
that region.This design region capability of PanelPlex can be used to, for example,
design to a bacterial target. Though bacteria are too big to design the
whole target because they’re millions of nucleotides long, but you can narrow your region down
to 40,000 nucleotides (or less), which is as big as any gene or gene region that just
about anyone would ever want to design to. And certainly, most viruses are shorter than
40,000 nucleotides. You could also use this to do a target in
the human genome. Lastly, we have to choose our hybridization
condition and I have one that I made up here for Zika. This is just a standard PCR condition in terms
of the sodium and magnesium that are present in the solution (this is your Master Mix composition). Lastly, you can assign modifications to your
probes such as TaqMan probes. I could choose Fluorescein at the 5’-end
fluorophor and BHQ Quencher at the 3’-end. That’s it. You hit ‘Run’ and the job is submitted. At this point, this program is going through
millions and millions of computations, and they’re being done on 32 CPUs. You can see the program automatically took
us to the results page and here is your job results. Here’s the project I just started, ‘Zika demo
two’. It says ‘Not Started’. It takes about 60 seconds or so to spin up
a dedicated 32 core instance on the cloud to solving your problem. It will crunch away on it until it creates
your solution. In the process, it’s folding all of those
sequence variants of the Zika virus. It’s trying tens of thousands of primer candidates. It’s doing hybridization reactions to those
Zika viruses. It’s checking for all of the false positives
against all the members of the Exclusivity set, the Dengue fever viruses, West Nile viruses,
all the other things you put in the exclusivity. It’s checking to make sure that those primers
don’t bind to the human genome.This job takes about eight hours to run and I ran one last
night. I started it at 4:00 PM and it was ready this
morning when I came in, and I wanted to show you the results. We can see here that the first solution it
found involves two primer sets, and I’ll highlight them here. Here are the two primer sets that it used
to cover all the 168 different sequences. You can see individually, the first set of
primers, which I’ll highlight. This first set of primers was able to cover
96% of the Zika variants. You can go into the details section, and the
software does show you which variants are covered by that primer set. What you can see now is the second set of
primers took the cumulative coverage from 96% to 100%. The second set of primers by themselves, shown
here, cover 85% of the Zika virus genomes. The two primers together (i.e. multiplexed),
have been designed to not interfere with each other and to completely solve the problem,
and have a cumulative coverage of 100%.I mentioned that we have a set of not just one solution
to the multiplex problem, but here’s a second solution where three primer set were required
to get a cumulative coverage of 100%. Here’s one solution with four different primer
sets that were required in the multiplex. Here’s one with three primer sets, et cetera. Here’s one more down here with two primer
sets that’s actually an excellent solution as well. if you’re a user who’s interested in knowing
what is it that is special about these designed oligos sequences. If you hit ‘View Details’, this is where all
that information that I shared with you about the folding of the DNA. Actually, folding of the RNA target, folding
of the amplicon, etc. We have a set of scoring metrics here that
are giving us the bimolecular folding energies, unimolecular folding energies. All that stuff is integrated into the scoring
equation.Everything is shown to you so you can see where these scores came from.You can
also look at the coverage here. If you want to see how well do these primers
cover the different targets, you hit ‘View Coverage’ link over here. It takes you to this page and it shows you
how the primers cover the Inclusivity set. You click over here, you can see how they
cover the exclusions that you don’t want it to cover, et cetera.In addition, all of the
detailed information you can also download as CSV files. For example, all this information about this
solution, you can just hit ‘Download Solution as CSV’ and voilà it will download that CSV
file. We can take a look at that file. The file goes to my “Downloads” folder. I can open up that file and it has all of
the sequence information. You can just cut and paste, and order those
oligos and test them out. If you want to order a particular solution,
you just copy across a row like that. Order those oligos, test them, you should
be good to go.

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