How to check breakpoint after detecting a CNV.

Question

[Fonso]

I have detected a CNV across 3 exons in a gene confirmed by qPCR. What are the next steps required to further confirm this? Do I need to do a blot? How do I go about sequencing the breakpoints?

[Fonso]

Thank you that's a nice diagram. I meant to say a CNV x2 was detected following NGS (ie a duplication in both alleles or triplication in on another allele...?). So what you are saying is that I need primers to the 5' and 3' end of the region in question? And is the reason for using cDNA to increase the efficacy of PCR?

[WouterDeCoster]

Please use ADD COMMENT or ADD REPLY to answer to previous reactions, as such this thread remains logically structured and easy to follow. I have now moved your reaction but as you can see it's not optimal. Adding an answer should only be used for providing a solution to the question asked.

[Fabio Marroni]

I am afraid I assumed a too simple situation. Your case is more complex and would need different approaches (maybe long reads?), but my answer is NOT applicable to your case (should I delete it?)

[WouterDeCoster]

The question is not very specific, so your answer is not wrong I believe.

Answer 1

[Fabio Marroni]

I assume that by CNV you mean a sequence of DNA that is present in 2 copies in some subjects and in 1 or 0 copies in other (i.e. a deletion). If by CNV you mean a portion of DNA that is present in multiple copies then the situation is more complex. This said, you might design a primer pair on the border of the CNV and confirm it either via PCR + gel, or by sequencing the breakpoint (Sanger would suffice). See the picture I created for guidance: basically, you have to design two primer pairs. If the 3 exon region is present in both alleles you will be able to amplify couple 1F-1R and 2F-2R but not 1F-2R. If your 3 exon region is heterozygous then you will be able to amplify all the three pairs. Finally, if you have an homozygous deletion you will only be able to amplify the pair 1F-2R.

[d-cameron]

Note that a CNV can be caused by more complex events that just simple duplication or deletions. For somatic SV in cancer, SVs are frequently caused my complex catastrophic rearrangements such as chromothripsis and chromoplexy. In such cases, paired flanking primers around the breakpoint are likely to fail and a more complex experimental design will be required.

[Fabio Marroni]

I agree with you, I assumed a too simplistic situation.