Question: Detecting Homozygous Insertion from Sanger Seq
0
gravatar for justinms
15 months ago by
justinms10
justinms10 wrote:

I have a patient with a homozygous insertion, although I can detect the presence of the insertion using the software that I have, it is not so clear as to whether or not the individual is homozygous for this insertion. Is there any way this can be detected using sanger data?

sanger indel • 945 views
ADD COMMENTlink modified 15 months ago by trausch1.4k • written 15 months ago by justinms10

I would look at the raw chromatogram and zoom to the insertion site. If it is heterozygous, you should see two signals coming up. Programs like SnapGeneViewer can visualize the .ab1 files. Heterozygosity would look like this:

Sa.ger

ADD REPLYlink modified 15 months ago • written 15 months ago by ATpoint25k

Correct. But this is a homozygous insertion and there would only be one peak at this site. I need to confirm that both alleles have the insertion

ADD REPLYlink written 15 months ago by justinms10

Well, if you see the additional nucleotides compared to the reference, and clean single peaks, then it should be homozygous.

ADD REPLYlink written 15 months ago by ATpoint25k
3
gravatar for rbagnall
15 months ago by
rbagnall1.4k
Australia
rbagnall1.4k wrote:

Yes.

  1. The Sanger sequencing trace will look very 'clean', with only single allele peaks on the chromatogram at each nucleotide (see the first 8 nucleotides of ATpoint's figure above), but there will be some additional bases compared to the reference at the site of the insertion.

Some other points worth noting:

  1. Increase the size of your PCR amplicon to include known common polymorphisms, and if you are lucky, one of them may show as heterozygous on your Sanger sequencing, confirming both alleles have amplified.

  2. Be careful that your PCR or sequencing primer does not anneal over a common polymorphism. This can result in you inadvertently amplifying only one allele - the one with the insertion - and it will look homozygous. This is called allele drop out, I think.

  3. If the insertion is rare, sequence the parents, as both may be heterozygous. (Although one or both parent may be homozygous if it is a common insertion).

ADD COMMENTlink written 15 months ago by rbagnall1.4k
1
gravatar for Andy
15 months ago by
Andy20
Andy20 wrote:

A heterozygous insertion will look like this;

sanger electropherogram

A homozygous one will look like a regular electropherogram.

ADD COMMENTlink written 15 months ago by Andy20
0
gravatar for trausch
15 months ago by
trausch1.4k
Germany
trausch1.4k wrote:

You can decompose the Sanger trace using Indigo. It should work for het. and hom. indels.

ADD COMMENTlink written 15 months ago by trausch1.4k
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