heterozygote with ngs and homozygote with sanger seq??

[avinash]

Dear friends,
I have a small problem interpreting an indel that I found using exome sequencing with Illumina GA IIx.
When I look at the exome sequencing data it seems to be a clear heterozygote. We used conventional Sanger sequencing to confirm the indel but found it to be a clear homozygote. Apart from contamination and sequencing errors could there be some biological reason behind this? I have confirmed that the reads do not match to any other region in the genome.

[krobison]

What is the frequency in the Illumina data of the allele you see in the Sanger data; Sanger (on uncloned material) has a significant sensitivity issue.

If it is really important, you might consider another assay such as a single base extension or padlock probe assay to determine what the frequencies are of the two.

[avinash]

What is the frequency in the Illumina data of the allele you see in the Sanger data; Sanger (on uncloned material) has a significant sensitivity issue.

If it is really important, you might consider another assay such as a single base extension or padlock probe assay to determine what the frequencies are of the two.

Thank you very much krobison for the suggestion.
I should have mentioned that the problem I mentioned above is from sequencing a single human DNA sample (for NGS and Sanger). So, I guess allele frequency should not be a problem (unless there is mosaicism).

[lh3]

Are you pcr+sanger? Are pcr primers affected by snps? Do you pcr genomic region or mrna?

[avinash]

Are you pcr+sanger? Are pcr primers affected by snps? Do you pcr genomic region or mrna?

If I understood you correctly, you mean do I PCR amplify before sequencing. No, not for Sanger sequencing. However the exome sequencing protocol does have some amplification steps.
I have checked for any SNPs at primer positions and there are none.
I am using genomic DNA for sequencing.

[lh3]

I know how exonome sequencing is done. I mainly referred to sanger sequencing. How do you sanger sequencing the region you are interested in?

[avinash]

I know how exonome sequencing is done. I mainly referred to sanger sequencing. How do you sanger sequencing the region you are interested in?

Oops!! I thought I was telling something that would help explain my problem better. Extremely sorry and disappointed to know that it sounded like I was teaching something.

[The_Roads]

Hi Avinash,

where are your sanger primers in relation to the exon and the indel? it may be that both results are true and that you are assembling psedogene sequence that is not amplifying with your sanger primers.

The_Roads