Microsatellite work has this problem. The best way to try and overcome it is to 'bin' your alleles into 2-3 bp repeat differences. For instance if you have an allele with 110, 111, 113 bp then you can group this as 1 allele 'bin' and your next 'bin' would be alleles of sizes 114, 115 and 116 etc. In terms of telling which is a 'true' allele from your electropherograms (I am not sure what you are studying- whether you expect one allele per sample or more) we always take the major allele (highest peak) per sample and any others that are at least 1/3 size the peak of major allele as 'true' alleles if you expect to have more than one genotype per sample. I hope this helps.

Its good to include a couple of positive controls spanning the range of your microsatelite size range with every run. This will help you keep consistent in your scoring. Also think about evaluating the whole group together rather than one sample at a time, this way you will get a feel for the frequency of the allele given HWE conditions. If its the only one allele of that size in a pop of 30 animals, you can probably confidently bin it with the more common + or -1 allele. If you can include more size standards closer to the target allele that can also help.

How was chosen this microsatellite? If you searched for sequences having 2bp repeats, in theory you shouldn't have a +1bp difference. In practice, you can have it in these cases:

- the reason explained by François

- the repeat is composite

- a misintepretation of stutter bands

possible cures:

- use a proofreading Taq polymerase

- sequence the supposed +1bp allele

- read the huge literature about reduction of stutter bands

Hi Bertha,

I had the same trouble some years ago when I was standardizing pcr multiplex in cattle. But there is an answer, As I can see, I am agree with Jacinta in one point, about the run conditions of the equipment you are using. If you realize, the first and the second sample were analyzed at 2012-08-02 but the third one was analyzed at 2012-09-10, another day, possibly, with another environment conditions. That probably affect the third sample elongating the signal of the EMU12 allele. So, the allele 154 really is the same 153 of the other samples.

One way to confirm this, is running the 3 samples at the same day, one after the other, you could see that these are the same allele.

I hope this can be helpful,

Hi Bertha,

I think allele calling is always confusing when we first started. Giving thought to how Taq works would be helpful. Sometime Taq adds an A to PCR product hence not all peaks are giving the true alleles size. This could be solved by binning at the size which majority samples possessed (ie bin at 153 if 80% of the samples have 153bp size, and 20% show 154bp). E.g. Make a 2-bp bin for all samples or covert those 154bp to 153bp manually, if the marker is a dinucleotide SSR. Also using a better quality Taq like Invitrogen’s Platinum Taq, although relatively costly compared to some others, would create lesser problems. Another way is to add control/ standard DNA on your plates, like what Ruth recommended. E.g. use, say, 3 or 4 samples acting as standard DNA and run them in all the plates for all markers. This is critical, especially when running samples on different days or different machines.

All the best for your genotyping work.

Hi everyone...

Thanks for your valuable information.

I'm sorry for late reply ;(

Kinda busy with my final project report.

And now, after consulting with my supervisor, then I already "re-bin" the alleles.

I also did the repeat two and try to determine the true allele by its shape.

Again, thanks for your advice :D

So helpful.

Best Regards

https://books.google.az/books?id=7Q_pCAAAQBAJ&pg=PA36&lpg=PA36&dq=How+we+determine+the+right+alleles,+when+we%27re+running+the+repeats+and+the+alleles+have+1+bp+difference?&source=bl&ots=91Gx3WR5Qj&sig=ACfU3U37vLsFgD30qQq7bSdyCbD6cUo6LQ&hl=ru&sa=X&ved=2ahUKEwjFm4aOqaTqAhVGwKQKHZq2AKAQ6AEwAnoECAkQAQ#v=onepage&q=How%20we%20determine%20the%20right%20alleles%2C%20when%20we're%20running%20the%20repeats%20and%20the%20alleles%20have%201%20bp%20difference%3F&f=false