AmpliSeq custom panel, target region sizes

[lethalfang]

Hi,

I want to look at ~ 100 point mutations in AmpliSeq in those very specific locations.
When I submit target regions, can I just submit single base locations (actually region sizes of 2, because the system requires that the end position be *greater* than the start position)?

Some people say I should have region size ~ 100, but I don't see the point for submitting region size of ~100 when I'm interested in only one.

Thanks.

[foofer]

Hi Lethal Fang,
Intimidating user ID. I actually have the same question. I'm chasing it down. If I get the answer I'll post it. I do know that even if you submit the 2 base region Ampliseq will design appropriate length amplicons (mine were around 100bp). Still I'm not sure if this is the best way to submit single base mutations.

[lethalfang]

Hi Lethal Fang,
Intimidating user ID. I actually have the same question. I'm chasing it down. If I get the answer I'll post it. I do know that even if you submit the 2 base region Ampliseq will design appropriate length amplicons (mine were around 100bp). Still I'm not sure if this is the best way to submit single base mutations.

I'm still not sure, either. But we ordered some anyway, and I'll see if the sequencing results are acceptable.

[Genomemirabilis]

You definetely need to specify the region. I´m just not sure if it´s the best strategy to perform the analysis.

For 100 point mutations, I´d look into qPCR unless you have a very small number of samples. With OpenArray plates the reagents costs would be a little bit higher than 20k for 1,000 samples (around $22 per sample).

[Genomemirabilis]

I´m also considering these point mutations are spread between several genes and they are not rare somatic mutations which would require the depth of NGS.

[foofer]

Entering in single base regions is fine for Ampliseq Designer.

I'm still not sure, either. But we ordered some anyway, and I'll see if the sequencing results are acceptable.

[lethalfang]

Entering in single base regions is fine for Ampliseq Designer.

Yes, I can confirm that. We sequenced some regions when my design is only 2-base long (only because the Custom design requires that the end position be greater than the start position, so I submitted 2-bp region instead of 1-bp single positions).
The results look alright.