Hi everyone,
I have one isolate that seems to be a new recombinant of a DNA virus. The problem is that genetic differences between major and minor parents are tiny.
Of course, my Sanger sequencing results might be untrustworthy, I'm planning to run cloning sequencing later on, but currently my PI insists that I should prove the feasibility of this recombination event in situ.
Are there any guidelines to prove that recombination event in certain sites is possible? I was suggested to build trees for each "chop" of my sequence and compare them with similar types of sequences but with rather high genetic differences, and for some reason to compare amino acid sequence and identify synonymous and nonsynonymous substitutes.
I'm wondering, is it helpful to identify methylation sites in situ and/or in vitro as well?
I'll be grateful for any piece of advice, any contribution.
Thanks.
Dmitry, have you thought about a simple diagnostic PCR approach with genotype-specific primers? Not always the most reliable, depending on your sequence and the difference between types but very quick and easy to do.
Otherwise with a little more effort you could design a RFLP digest assay which is often possible even with minor differences in sequence. Free restriction mappers are available online, eg. http://www.restrictionmapper.org/
5-7 years ago, I used Simplot to show event of recombination. Link: http://sray.med.som.jhmi.edu/SCRoftware/simplot/
Import your alignment file (in fasta format), go bootscan, under command tab choose your query group/ seq. And start the run.
And now it seems that SSE is better version for the analysis. http://www.biomedcentral.com/1756-0500/5/50
You will be able to identified where the recombination happen (if there is any), then U can run trees using only partial sequences (before and after the point of recombination). Your candidate should cluster with different group using the partial sequence.
On what basis you are considering as a recombinant? Look at your sanger sequence raw data carefully. If you are getting mixed peaks there are chances of infection with two or more pure genotype/subtype also known as co-infection/superinfection. Cloning will help you in this case or if there are more than one type of recombinants in quasi-species.
I agree with Chan Shie Yien, I have used SimPlot for the recombination detection. You need to take panel of sequences different subtypes/pure isolates and then use the SimPlot. After getting breakpints you can make phylogenetic trees with those fragments.
Using genotype specific primers may be an option but you can't get break points. recombination can be at any place in the entire genome.
You can refer attached publications:
If you need further help, contact me: [email protected]
Sudhanshu Pandey
orcid.org/0000-0002-9843-9809
SimPlot can be used provided you have parent and recombinant sequeces. Other programs that are useful are GARD and SBP from DataMonkey
http://checkforplagiarism.net/
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