Hi, I'm a beginner in the SSRs fragment analysis. I am using some di- and trinucleotide SSR primers. I expected that the allele sizes it could be similar respect the expected size reported in the table sheet of the marker. But GeneMapper software detected different sizes. In the attached file, two examples of my results about sample 01 and sample 09.
Information of the markers:
Marker Pie 239 (AT) dinu-, repeat number 12, expected size (bp) 95
Marker Pie 227 (TGG) tri-, repeat number 8, expected size (bp) 160
Marker Pie 223 (GGT) tri-, repeat number 8, expected size (bp) 200
Marker Pie 215 (GAC) tri-, repeat number 6, expected size (bp) 200
I would know also if there is a guide for the interpretations of the result in GeneMapper.
Thank you very much!
Dear Antonio Luca Conte,
In my previous experience with SSR's, I would like to Suggest you to use "GeneMarker" Software. If your all data files are ready for analysis, then you can download "GeneMarker" software, free trail for 35-90days, which maybe enough for your data analaysis. Furthermore, I would like to suggest you my 2017 SSR's research article and 2018 SSR's review article, I am sure you will get clear idea about, how to analysis these peaks in GeneMapper/GeneMarker software (Please find the attached files). Here in my case "Study on Sugarcane" Generally speaking, the presence of a peak can be scored as an allele in diploid plant species. However, in sugarcane, peaks of different heights or shapes often affect their correct scoring and data analysts. As such, one often wonders if a low height peak or a minus-A peak can be scored as a new allele. Therefore, different analysts may produce different scoring data from the same sugarcane genotyping files. The size of any allele estimated not only depends on the number of nucleotides, but also on the mobility of the fragment in the electrophoresis, the type of fluorescent label, the distance of the allele from the size marker used, the DNA sequencer, and genotyping software. Furthermore, I will guide you, how to analysis your data?.
Dear Ali, thank you very much your paper is very usefull for me. Bye
Hi Ali, I have a doubt, I have different alleles in size 156 (Primer 227, see the image above) with a pink band, it means peaks off-ladder. I can use that allele if there aren't overlapping with other peaks? Or when I have off-ladder I have to repeat the sequencing or PCR?
Thank you ...
Dear Antonio,
For these situations, you had better to check your data on multiple Software and compare, what differences you got. or First sit your software programming according to each primer pair size. Sometimes these peaks accuracy also depends on the florescent dyes usage.
Now coming to your GeneScan file image, your first image shows only one correct peak (Blue) the other may not be considered during reading. Still you had better to re-sit your software programming before running any primer SSR's data. Please find the attached files, which will shows you, how to read the correct SSR's alleles.
Ok, and in the second image, the only one peak (Blu) in Primer 227, can I consider it good?
Thank you very much for your advice.
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