difference in sequencing results - (Nov/18/2007 )
Dear all,
I have this little problem where a piece of sequence amplify by two different kit..... giving me two different sequencing results.
I want to sequence a structural gene of a RNA virus genome:
First reaction amplify using Roche kit and second reacction amplify using Qiagen kt......
Both gave very clean sequencing results..... but both results are contradicting in their viral properties.
Any suggestion..??
Thanks
It's not clear what you have done. Did you do an RT- PCR reaction? What was the enzyme? What were the primers? Were they the same? How did you clean the reactions up? What primers did you use for second round cDNA amplification? What primers did you use for sequencing?
We can't intelligently answer questions unless we know what you did.
We can't intelligently answer questions unless we know what you did.
Dear Phage,
Here I am comparing the sequencing result obtained from two different kit......Generally the work flow are as such:
RNA extraction from plasma --> RT-PCR reaction --> second PCR --> PCR clean up --> sequencing reaction.
RNA extracted using QIAgen viral RNA mini kit....
RT-PCR using the same primer.... for both kit
second PCR also the same primer.... for both kit
PCR clean up also QIAgen....
and the sequencing service provider also the same.
The only thing different here is one I amplify using Roche one-step high fediity RT-PCR kit and another one using QIAgen one step RT-PCR kit.
and when the sequencing result come back, Roche and QIAgen shows different result..... which one I should trust?
Thanks
Did you run the second PCR reaction on a gel? Was the length of the fragment the same in both cases? Did you purify the PCR fragment from a gel? What primer did you use for sequencing? Have you blasted the two results to see if they are similar to one another, or to known sequence?
Dear Phage,
1. Did you run the second PCR reaction on a gel?
Yes I did run second PCR product on gel.
2. Was the length of the fragment the same in both cases?
Both PCR product from ROCHE and Qiagen show the same size.... and there is only one band.
3. Did you purify the PCR fragment from a gel?
I did not purify from gel.... i took the remaining PCR product and subjected to PCR clean-up kit.
4. What primer did you use for sequencing?
The first PCR, second PCR and sequencing reaction were using the same primer.
5. Have you blasted the two results to see if they are similar to one another, or to known sequence?
I align the two sequences..... and it shows > 95% similarity
however when I analyse them, Roche one classify as wild type whereas Qiagen one classify as mutant.....
My problem is:
I am wandering why same PCR product derived from same RNA extraction can give different PCR results?
Which one I should follow....?
What should I do now? Can I send them to another service provider..... but i worry that another service provider give another kind of result that is even more confusing me.....
sorry for giving you this kind of problem
Thanks
Tough one. If your results are so clear, you'd have to assume that the results are true and original sequences were different. Did you use different clones of the virus (or whatever you call viruses)? Different clones can contain variation even if they are the same viral species (or whatever you call viruses).
Yes... we knew that virus has quasispecies, especially true for RNA virus.
However, if that is the case, I expect to see double peaks on the same position (sequencing result).....
rather than 2 nice single, clear, nice, no background peak on two different sequencing results.
Any explanation?
Best regards
Oche
What was the viral load? Is it possible that you were near the detetion limit and amplified a different clone from the quasispecies?
Was it the exact same sample you amplified by RT-PCR (same RNA extraction) or different samples from the same patient? (or cell culture experiment)
In your amplification: were your negative controls negative? Do you more often work with this wild type (possible source of contamination)?
How is the classification done? Perhaps the differences are quite minor, but are enough to transition from wild-type to mutant in a black/white kind of evaluation. Are you removing questionable bases from the sequencing results? The first 40 bp are usually trash, and anything after 800 bp is questionable. There are also often dye blobs around 80-120 bp or so, which can be read incorrectly by automatic software.