two different scaffold which were connected in real genome. how can I verify?

Question

[jth6974]

Yesterday I asked about same question and I got answer like below

"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." by jrj.healey

but I still didn't understand 100%.

I need an example about it.

1.Let's say there is a DNA sequence like,

5' ACCTAGCTGG 3'

2.And After NGS and genome assembly I got two scaffolds like,

scaffold1 : ACCTA scaffold2 : GCTGG

Can anyone who can explain with that?

Answer 1

[Joe]

Those sequences are too short to serve as an example, and there is no missing sequence in your example which is the whole point of the exercise.

Imagine you have the 3’ end of Contig A:

...ATCGACTAGCTAGCATCGATCGACTAGCTACG

And the 5’ end of Contig B:

GTACGATCGTAGCGGGCGCATTATCGG...

For this example, we will assume A is ‘left’ (5’) to B in the actual genome.

You would design primers which ‘aim’ at the gap between the 2 contigs.

             Contig A.                                  Contig B
—-ATCGACTAGCTAGCATCGATCGACTAGCTACG.............GTACGATCGTAGCGGGCGCATTATCGG—-
                                  ^           ^
                  Unknown sequence in the ‘join’ between contigs

Primers would be designed like so:

     Forward primer  TCGACTAGCTACG —->
—-ATCGACTAGCTAGCATCGATCGACTAGCTACG.............GTACGATCGTAGCGGGCGCATTATCGG—-
                                           <—- CGCTACGATCGTAC Reverse primer 
                                                             (reverse complemented)

PCR using those primers from a gDNA template would hopefully yield a fragment that you could subsequently sequence with Sanger sequencing.

I would advise you to search google for papers and figures with the query “gap closing by PCR” or similar.

[jth6974]

Thanks to you now I totally understood !! really thanks for your kindness~!!