35407 I would store the data as read from the electropherogram. On analysis round up/down the major peak in the usual arithmetic >0.5 up and

Interpreting fragment analysis results can be challenging, especially when dealing with novel SSRs. Here are some suggestions that might be helpful:

In addition, it might be helpful to consult with other researchers who have worked with Clivia miniata SSRs or to search for relevant literature to obtain additional information or guidance. Good luck with your research!

In terms of resources, there are many articles and resources available online that can help with SSR marker development and interpretation. Here are a few examples:

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587563/
• https://www.frontiersin.org/articles/10.3389/fpls.2017.00206/full
• https://www.researchgate.net/publication/341258888_Development_and_evaluation_of_new_SSR_markers_in_Clivia_miniata_and_their_transferability_in_other_Amaryllidaceae_species

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

Thank you Nikolay Klyashtornyy and Paul Rutland for your help.

I ended up using the general trend of my data. Considering locus 63662, which had potential amplification around the 121/122 region as well as around the 319bp region. I repeated the PCR and decreased the number of cycles which gave amplification around the 121/122 region. Since this was a dinucleotide, I considered alleles that were either 2 bp apart or multiples of two. In this case I had possibilities of 121/131/137 or 122/124/128. I ended up deciding on the 121/131/131 repeat pattern as it was throughout more than 80% of my result. With the 122/134/138 pattern, repetition of some of those scores gave the 121/131/137 trend. As Paul Rutland suggested it might've been due to enzyme adding an A base, repetition of some of the reactions somewhat helped with understanding this. Nikolay Klyashtornyy suggested checking primer specificity. The 319bp amplification was not seen throughout the samples. Only a few had this type of amplification. Decreasing the number of cycles helped (I also diluted my DNA to ~20ng/ul).

Regarding 3 by Nikolay Klyashtornyy , I ran all my PCRs with a sample of known allele scores. This helped a great deal as it helps with some results which didn't make sense. As I would know based on the reference if there was any issue with the reaction itself.

I also repeated the reactions for 2156. I did separate reactions whereby I changed one parameter at a time (i.e., number of cycles or annealing temperature). A higher annealing temperature and decrease in the number of cycles made the results a bit clearer. Most of the other peaks were removed, 206/208 was the resulting alleles I ended up using. They were more consistent and RFUs were higher.