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CONTEXT


I am currently working on a set of diversity, this diversity in interspecific (within the same genus). I am using SSR markers, the primers were designed on one species and are working really well within the species (lot of diversity and difference between allele of 1 at least).
When trying them on the other species, I observe the normal non-amplification which could be due to mutation in the primer DNA sequence. But, for some genotypes I have been observing a new allele just between the two older ones (like the new one is 185.5 and the olders were 185 and 186.

QUESTIONS


Is that kind of observation frequent when using primers designed on other species?
I understand how a 1 pb difference could occur CACACA ==> CACCACA, but what could the biological explanations be for a 0.5 pb difference between alleles?

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In my experience, working with a similar approach in Campylobacter jejuni, the base pair measurements from these techniques are imprecise and need to be carefully calibrated. I am unsurprised to see 0.5 base pair differences, this can be seen been runs and even, in the worse case situation, between even and odd wells in the same plate(!).

I would use Sanger sequencing to determine the sequence you are getting in one of your isolates, and then use this as a calibration control included in every run.

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  • $\begingroup$ Thanks for your feedback! I was planning to do a sanger sequencing to understand the problem. The allele though is quite stable (same length on different plates). Usually I see all my alleles in a 0.5 pb variation around the real value depending on genotypes/runs. $\endgroup$ – Untitpoi Jun 20 '18 at 11:31
  • $\begingroup$ @Untitpoi We found that different length targets from the same strain, and the same targets from different strains frequently gave different length results. $\endgroup$ – Jack Aidley Jun 20 '18 at 12:18
  • $\begingroup$ @Untitpoi I think it occurs because of differences in the sequence causing sligtht differences in mobility during capillary electrophoresis. If you look at the raw data from a sequencing read, you can see that the peaks appear less regularly than the smoothed processed peak data you usually look at. I would guess there are consistent differences in the progress of sequences based on the physical properties of the nucleotides. $\endgroup$ – Jack Aidley Jun 20 '18 at 12:28

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