How to validate the deletions found in Golden Standard, but not in ngs/3gs?

Question

[cholingken]

Hi all,

We are doing a study of Golden Standard of structural variation for NA12878 (http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase3/integrated_sv_map/). We found that there are quite many large heterozygous deletions(>10kb-200kb) in Golden Standard result, but no NGS nor pacbio/nanopore reads support that, anyway to validate these deletion by wet lab?

Thank you very much!

Answer 1

[finswimmer]

Hello,

the first thing that came to my mind is to validate it with MLPA. For deletions >50kb array CGH might be an option. If you know the breakpoints of the deletion, you can do it also by PCR. At least the smaller allele (the one with the deletion) could be amplified. Afterwards you can sequence it by sanger.

fin swimmer

[cholingken]

Thanks Fin,

We know the breakpoints and have had a plan as showed below, please comment.

Designed primers for each of them, and then would do Sanger sequencing. Here are our expected results (assuming the primer is highly specific): 1.if there was really a deletion(homozygous), no PCR product/band. 2.if there was no deletion(homozygous), PCR product/band. 3. However, things become complicated if the deletion was heterozygous, which means there would have PCR products. This will make us think that there is no deletion.

Answer 2

[finswimmer]

Hello again,

if you know the breakpoint, than you can take one of these approaches:

1. The Quick&Dirty Method:

Make two seperate PCRs.

PCR 1 containes a Primer pair where one Primer is located before the first breakpoint and the second primer is located within the deleted region. You will only get a product if you have at least one allel with no deletion.

PCR 2 containes a primer pair where one prime is located before the first breakpoint and the second primer is located after the second breakpoint. You will only get a product if you have at least one allel with a deletion.

In summary:

• PCR 1 ok, PCR 2 failed --> no deletion homozygous
• PCR 1 ok, PCR 2 ok --> deletion heterozygous
• PCR 1 failed, PCR 2 ok --> deletion homozygous

Of course the PCR can fail for other reasons.

1. The more elegant way: Multiplex PCR

You need four primers:

• F1: Forward Primer before breakpoint 1
• R1: Reverse Primer after breakpoint 1
• F2: Forward Primer before breakpoint 2
• R2: Reverse Primer after breakpoint 2

F1/R1 and F2/R2 will give a product if there is an allel without deletion. F1/R2 will give a product if there is an allele with a deletion.

So if you have after PCR:

• 2 products --> no deletion homozygous
• 1 product --> deletion homozygous
• 3 products --> deletion heterozygous

The difficulty in this method is to find primers with similiar annealing temperatur. And the possible products must have a difference in size that can be seen in a gel electrophoresis.

fin swimmer

[cholingken]

Hello Fin,

Thank you very much for your effort. Your information is very helpful. However, for the deletions we are studying, the size ranges from 10kbp to 200kbp. Do you know which polymerase can do that? And the primer design for the multiplex approach would be very challenging.

Ken

[finswimmer]

Hello Ken,

there is no need to amplify the whole region of your deletion. Maybe the trick becomes clearer with this:

• F1, R1, F2, R2 are the primer I described above
• "-" is your DNA
• "|" are the breakpoints

-----F1 >--------|-------< R1-------------10kb-------------------F2 >----------|---< R2----

In case one allele have no deletion your amplified products look like this:

• F1 >--------|-------< R1
• F2 >----------|---< R2

The region from F1 to R2 want be amplified, because the distance is to great.

In the case there is a deletion F1/R1 and F2/R2 cannot be amplified because the primers R1 and F2 cannot bind anywhere. Instead F1 and R2 come closer together and the region between can be amplified successfully:

• F1 >-----------< R2

fin swimmer