I am using developed markers from a closely related subspecies (stem rust fungi) and have 24 markers that are working well in 3 host species, all amplifying in the correct patterns expected (e.g. trinucleotides with allele sizes 103, 106, 112, etc.). I was asked in a lab meeting if I assume when I see products of the same size in different samples on capillary electrophoresis if I assume that they are the same product. The answer is clearly, yes. I don't know of anyone who would spend the time to sequence each and every product from population studies of hundreds of individuals or if that is even remotely a necessity. But, I have not sequenced these products at all, and I am just assuming that based on size and repeat patterns (6-8 alleles separated by the correct number of base pairs) that they are the right products. How much assumtion is ok and how much verification is expected for using these markers?
Hi Vicky,
quick disclaimer: I assume you are doing population genetics within a single species. I don't know of anyone who sequenced STRs just to check this either. Most of the commonly used STR loci are also well known and have proven their usefullness in countless other studies.
Generally spoken, the main reason for STR is their mulit-allelic nature and fast mutation rate. However, there is of course always a saturation problem as well. Given allele 150 mutates and new allele 153 (trinucleotide) is created and now present in the population. Whenever allele 153 mutates through slippage f.e. (presumably following the stepwise mutation model, which is a common one for STR - but you might want to look into that further), then the new allele could either be 156, or 150 again. In the second case, we would wrongly assume that individuals harbouring the "new 150" allele do have a lot in common with individuals with the "old 150" allele, which they actually dont (as there are two mutational steps in between them).
Given the nature of mutation in STRs (probably adding/loosing a repeat motif per step), I wouldnt be sure if you would catch the "new allele 150" and the "old allele 150" as being different... their sequences could just look alike.
Then again, in population genetics we work mostly with allele frequencies, on population or subpopulation levels. Having several loci and each of them being polymorphic for a good number of alleles, I would assume the bias described above should not be that drastic.
Cheers, Florian
EDIT: formatting
Hi Vicky,
If you are doing population genetics, where you try to differentiate between strains of one particular species, you shouldn't need to sequence your products (for which you would probably need to desgin some specific sequencing primers anyways, as the flanking sequences next to the STR region could be only a few basepairs). However, it all depends on previous studies performed with the markers you are using.
During development of the STR assay, the markers should have been tested on a wide selection of isolates, and PCR products of at least some of them should have been sequenced. If the typing assay was validated correctly, you shouldn't need to sequence the products yourself anymore, because it has been done in the past for all the markers. Of course if it's a really old assay, you could sequence a handful to get more certainty.
A huge advantage of STR typing compared to good old sequencing is that it's way less time consuming. If you have to sequence all your PCR products anyway, you might as well do MLST, or WGS SNP analysis if you have the funds.
Good luck with your research, Merlijn
Hi Vicky,
I guess the answer depends on the organism you are dealing with and its evolutionary history.
In my case, I was working with an ancient tropical root crop (Colocasia esculenta) for which at least three centers of domestication have been identified, that has diploid and triploid forms, two chloroplast morphotypes, and several phyletic lines. However, at the time I did not know that, I just thought I was dealing with one single entity. SSRs had been just published and tried on some samples from the Pacific, whereas I was working with samples of African origin and comparing them with Asian/Pacific ones.
In a first screening, the electropherograms were showing a single band roughly of the same size, but since I was screening hundreds of specimens, sometimes I would see a slight difference of a base or two. It wasn't clear. So eventually, I decided to sequence some of the individuals for which I had doubts (and then all of them) and sure enough, I ended up with six very different allelic forms. What looked like a monomorphic locus, with products of the same size, was actually a polymorphic region, and if I had just screened some individuals, maybe I would have never seen all the allelic forms that I saw. That said, mine was an interesting but probably not very common situation.
Hope this helps,
Ilaria
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