how do i analyse cloned sequences - Clone and DNA sequence (Jul/26/2005 )

Hello all,
I sequenced few clones. At some nucleotide base positions I find that one clone has "G" sequenced in either direction and for all other clones at the same position is a "T". I dont know how to interpret this. What would be the issue? I have this type of sequence read at multiple positions in my sequence.
Thanks,
Solution

>>At some nucleotide base positions I find that one clone has "G" sequenced in either direction and for all other clones at the same position is a "T".

It could be a SNP. If all clones are derived from one DNA sample, this sample is heterozygous G/T for this SNP.

Hi solution
How did you obtain the clones? If you did RT-PCR there could have been errors introduced in one clone that are not present in others... It could also be sequencing error, especially if all of your differences are in the same clone, it may have been a bad run. If you can get it, a print-out of your sequence run showing the peaks for each base would help you decide.

Hi solution,

beccaf22 is right, first of all, you must check your chromatograms. It could be sequencing errors.


DNA Sequencing TroubleShooting Guide :

http://www.genome-express.com/site_gex_by_...GEX_04_2003.pdf

Both beccaf22 and pcrman are correct!

Thanks for your response.
I obtained the clones from PCR amplification and then plasmid transformation. A thing about the DNA is that it is from a virus, so how can it be heterozygous when the virus just has one circular DS DNA?
The chromatograms are clean in those regions. For your reference I have attached part of the sequence of the chromatogram.


Thanks,
Solution

Hi again,

The sequence does look good, so my next guess is the PCR introducing errors, even with high-fidelity taq there is a chance of introducing mutations...

In this case you would expect that different clones will have mutations in different places so if you clone the same sequence say 6 times and 3 are G and 3 are T (ie: the T nucleotide at the same position is present in multiple clones and the G nucleotide is present at that position in multiple other clones) then I would lean toward the SNP hyp. by pcrman.

If each clone has one or two bases different from all the other 6 clones then I would think pcr errors and take the nucleotide at that position in the majority of clones as the "true" sequence.

Howdy,

Great suggestions above, but here's just another one (hey, I'm a virologist!)

Since it's a viral sequence I'd also check that the differences aren't in a "crucial" part of the genome (that is, of course, if the sequence has previously been determined, or you have more information about the virus). Check to see if it introduces any stop codons, or if the changes alter the protein. For example, if it produces a non-functional polymerase, and you know that your virus replicates, you can pretty much determine which sequence is accurate for the viable virus.

I sequenced an RNA virus for my PhD and found that there were some changes that were consistently inconsistent! Don't forget, part of the beauty of viruses is that they are so variable, so you may be seeing true variation in your viral genome. It won't be a SNP in the sense that most people see it, since you will have many many many more copies of your starting material, all of which have been independantly replicated, cloned and sequenced.

Hope this helps!

Nicole

Agree with the fact that virusses are variable. Even if you have a DsDNA-virus, not every single virus from the same batch are exactly the same (not even all cells in my own body have exactly the same DNA sequence, that's the origin of evolution). Also, it might very well be that it's PCR induced mutations you're seeing. Check the protein sequences (if it's in a protein coding sequence of course).

Thank you all for your replies. To add, I get the variation in the sequence for the same clone of the virus, that is to say, if i have 7 primers amplifying a region 4 primers give say G the others give me a C, in addition some primers indicate that there is insertion and others primers for the same region do not have those bases and hence do not show that insertion. I m really confused.
Plz help.
Thanks