ViroSeq analysis problem

[Ivoalexiev]

Hi Guys,

I am new to this forum and I am honored to meet you.

I work in HIV resistance diagnostic and I am new to ABI sequencing.

My problem!
I use ViroSeq kit to analyse HIV PR and RT pol gen sequence. I have good sequences but forgot to add double underscore before the primers definition and ViroSeq software can not combine and align overlapping primers (sequences).

Best.
Ivo.

[Ivoalexiev]

Hi Guys,

I am new to this forum and I am honored to meet you.

I work in HIV resistance diagnostic and I am new to ABI sequencing.

My problem!
I use ViroSeq kit to analyse HIV PR and RT pol gen sequence. I have good sequences but forgot to add double underscore before the primers definition and ViroSeq software can not combine and align overlapping primers (sequences).

Best.
Ivo.

Hi there I found solution, If somebody have this problem I can help

[mohd2b]

ViroSeq sequencing

hi Ivoalexiev

Good to know that you are using ViroSeq for resistance studies. Iam also using it. however we facing problem with getting good and clean sequence.

Is it possible to share your methods of Quantification and cycle sequence product purification. May be we can adopt the method u use.

Are following Viroseq protocol or you have made some modification?

Waiting for your reply

thanks

[Ivoalexiev]

Hi mohd2b,
I use 16 capillary ABI 3130xl analyser. I see that you work in a hot country, primarily in the laboratory we like to keep temperature below 20 degrees, I keep 18. I follow the ViroSeq HIV-1 protocol - Genotyping System v2.0 and recently I started with the new analysis software - ViroSeq HIV-1 Genotyping Software v3.0. Following PCR purification I use 2 instead of 3 ul PCR Cleanup Reagent and after the Cycle Sequencing I use Ethanol/Sodium Acetate and I add 2 ul EDTA 125 mM. Also after the Hi-Di Formamide I go for denaturation - 93 degree for 2 minutes. Also you can check the POP, I use POP7 it have some advantages compared to POP6. Be sure you are using fresh and properly stored POP and capillaries.
Hope this help.
Ivo

[mohd2b]

Viroseq sequencing

hi Ivoalexiev

Well so far we find out that we have problem in measuring the right concentration of the PCR purified products, which is very critical in cycle sequencing reactions . Unfortunately, we do not have quantification software, instead we use ViroSeq gel quantification method. how do you measure the concentration? do u use Nanodrop??

also we have tried to use sodium acetate , can you share your Sodium Acetate protocol ? what other purification method you use in the lab other than SA?

when you analyze your sequence , what is the best range for raw data signal intensity that work as threshold ??

Thanks
Mohammed