how do i analyse cloned sequences - Clone and DNA sequence (Jul/26/2005 )
Hello Nicole thanx for ur reply,
Can u explain ...i didnt really understand when you said that :
there will many many many more copies of your starting material, all of which have been independantly replicated, cloned and sequenced.
Would that mean that each clone could have a different sequence and that it should be interpreted as the variation in the viral sequence and not as the PCR/sequencing artifacts? I have to make a report on the variation and intrepret and present it, I would appreciate ur help.
What I mean is: how do you know you have started with only one viral template for the PCR? If you have started with only one copy then the variation would be due to the PCR or sequencing error. But getting one copy of the virus is virtually impossible. I'm not saying that the sequence *has* to be due to a real variation in virus sequence, but if you have tried to eliminate Taq errors (using proof-reading DNA polymerase) and sequencing errors (by sequencing the clone more than once in both directions) then it MAY be that the difference you are observing is real.
Have you checked to see if any of the proteins are modified, by looking at open reading frames? One of the best programs I've found for this for virus ORF prediction is Frameplot (http://www.nih.go.jp/~jun/cgi-bin/frameplot.pl) - It shows you all 6 reading frames and all possible ORFs. Use this for all your different sequences and if any look really bad (for example, no ORFs) then you can suspect that the sequence is flawed.
If your virus already has some sequence info avalable (or is similar enough to another virus), you can align them and see if there are any obvious flaws in your sequencing.
Good luck with the report - the more info you can present saying that you have followed up on the leads (both experimentally, bio-informatically and logically), the better.
Nicole (glad to know that being a virologist is useful!)
Thank you all for your replies. It really helped. Thanks Nicole!