There are different primers used in sequencing of D Loop region of mitochondrial DNA in horse. Will different primers not result in different sequences? and Can the sequences obtained using different primers in different individual still employed for phylogenetic studies?
Hi Jigme,
If your primers have been successfully used to amplify the D-loop of the species you are studying, I would suggest you pick just one pair. Instead of using multiple primers for one gene region, why not get additional primers for other gene regions such as cyt b and co1. This will make more sense for a better comparative phylogenetic study.
Best of luck
Mark
Dear Jigme Dorji,
If you are able to obtain mtDNA D-loop sequences of horses using your primer pair or pairs then you can use this data together with available mtDNA sequence data in GenBank (provided by other researchers). Consequently, your data could be used for phylogenetic studies.
Kind regards
hello
if you are able to obtain mtDNA D-loop sequences of horses using your primer pair or pairs then you can use this data together with available mtDNA sequence data in GenBank (provided by other researchers). Consequently, your data could be used for phylogenetic studies.
They should all generate fragments from the same Dloop sequence unless they amplify different regions that do not overlap. If you have overlapping regions that do not match within an individual then this is a problem and probably indicates that you are amplifying a NUMT, which may be hard to tell since it is non-coding. If the regions do not overlap and you want to combine them but do not have a complete dataset for each region (i.e. different individuals have different fragments with some having both fragments) then you can run a supertree analysis.
My suggestion is to download a complete Dloop sequence from GenBank for the closest relative you can find and align your sequences to the reference sequence. If the fragments you have overlap but are very different then you should run away since one or both are probably NUMTs and you can not really tell with Dloop since it is non-coding. If they do overlap and are identical within an individual then you can join them together and run a standard phylogenetic analysis. If they do not overlap then you can either concatenate the datasets and run a standard phylogenetic analysis and/or run a supertree analysis.
Hope this helps.
Best Regards
Dear Max,
If I elaborate my case, it is like what you suggested. we have sequenced D-Loop (613 bp) of 26 Bhutanese local horses. The sequences obtained were aligned and further reference sequences (>600 bp) from other horse breeds were obtained from the Genbank. The sequences were aligned and sorted using G-Block software. The sequences in our study and those from Genbank overlapped to yield sequences of about 512 bp in all cases. In this case, what can we say about NUMTS? Will NUMTS be taken care during alignment and g-block sorting? and can I proceed with phylogenetic analysis?
Thanks
Hello Jigme,
From that you can not really tell much about NUMTs. Do you have a reason for thinking it may be NUMTs?
If the sequences seem reasonably similar to what you expected and you feel that they are actually mitochondrial Dloop then you can just use the 512bps that overlap for your phylogenetic analysis. You could also try the whole alignment to see the impact of missing data on your analysis.
Best Regards
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