How can I check if two scaffolds were neighbored in genome using PCR?

Question

[jth6974]

After finishing genome assembly I got a lot of scaffolds. and I found two scaffolds that were supposed to be connected. So I want to verify if those scaffolds are neighbored in genome. When I ask my professor, he said that I can check using PCR and if two scaffolds are got connected using PCR it means those were actually neighbored in genome. But I didn't understand at all cause I'm just doing computational work in my lab. Is there anyone who can explain about it?

[Joe]

If 2 sequences originate next to one another in the genome, you can PCR using primers that face toward the junction to amplify the missing sequence. When you sequence that via Sanger sequencing, if the fragment overlaps both contigs, they are adjacent in the real genome.

This only works if the missing sequence is short enough though.

[jth6974]

thank you !! you mean before PCR for filling the gap, do I have to be sure about those two sequences are adjacent in the real genome in advance? what should I do if I'm not sure about the order of sequences?

[Joe]

You don’t actually need to perform a PCR for the approach to work. For most Sanger sequencing services, you can simply mix some template DNA with a single primer, and send that away. You’ll get about 1kb of sequence back (hence what I said about the gap being small enough). You dont need to know the intervening sequence therefore, and just need primers that match the ends of your known sequences.

However, its likely the gaps will be large, and you will have to perform ‘primer walking’ by successively sequencing, designing a new primer for the new sequence, and on and on.

A much less labour intensive process would be to perform some long read sequencing of your genome with Nanopore or PacBio (though this will cost more money) and create a hybrid assembly.

Forcing anyone to do primer walking in this day and age feels like abuse to me :P

[jth6974]

what is DNA template corresponding to gaps???

Answer 1

[ATpoint]

PCR is a method to produce copies from a template piece of DNA. To amplify a given piece of DNA one requires a little DNA oligonucleotide called a primer that binds the single-stranded template and allows the enzyme that does the actual amplification (polymerase) to bind. In the below picture the template is blue, the primers are yellow and purple and the polymerase would start amplifying at these yellow and purple sequences to eventually produce a double-stranded piece of DNA as seen in step 3. One requires both a forward (yellow) and reverse (purple) primer for a successful PCR amplification.

In your case, one would design a primer pair with the forward on scaffold A and the reverse on scaffold B in a way that the product would have a resonable size, e.g. like a few kilobases or so because that is still in a range that can be well amplified. Conventional PCR works up to a few kilobases. Longer products are also possible and require long-range PCR. In any case, if the two scaffolds are not in proximity, you would not get a PCR product because, as said above, a successful PCR requires that both the forward and reverse primer sucessfully amplify the template sequence. If you never heard of PCR, it is a bit difficult to get your head around the concept, but there is a lot of online material available on it. As jrj.healey said, once you have a PCR product, you could send this to Sanger sequencing in order to confirm that it indeed comes from the two scaffolds.

Answer 2

[jth6974]

Even if I design primers that face toward the junction, amplification won't happen cause there is nothing between two sequences yet right?

[ATpoint]

What do you mean by yet?

[jth6974]

what is DNA template corresponding to missing sequence between two scaffolds?

[Joe]

That’s what you’re trying to find out...

You seem to think that that sequence doesn’t exist. That’s not the case, there is just unknown sequence there that didn’t come out in your assembly.