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aakechin / NGS-PrimerPlex

NGS-PrimerPlex is a high-throughput tool for mupltiplex primer design
GNU General Public License v3.0
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Intern Amplicons length does not overlap with amplicon length in info.xlsx #27

Closed marcellosalvi closed 2 years ago

marcellosalvi commented 2 years ago

Dear @aakechin I'm struggling in understanding what the programm mean for the internal amplicon file. I expected each sequence of 160 bp but I found more than 360 bp per amplicon. Could you explain me how is it possible?

aakechin commented 2 years ago

Dear @marcellosalvi Could you please write here the log file? There will be all the parameters that you used.

marcellosalvi commented 2 years ago

Thank you for your answer, I attached the log file. I set the optimal amplicon length at 160 bp, while the internal amplicon.fa has an average length of 330.

list_cds.log

aakechin commented 2 years ago

@marcellosalvi, I understood the problem. I remembered that the internal amplicon file contains internal amplicon sequences plus minus 100-150 bp to have an opportunity to move primer if it is necessary.

marcellosalvi commented 2 years ago

Thank you @aakechin. So in order to allign them to check the coverage I have to remove the extra bp. Are they 100 or 150? On ehich depends? ANd I would like to ask you also an other question: How do you choose the the primers? I see that the software produce ten different set of primer, which one I have to pick up?

aakechin commented 2 years ago

@marcellosalvi -100 and +100. Actually, many primers may be suitable, however, they are chosen based on the parameters that a user applies in the command and of course partially randomly among primers with equivalent parameters. So, you can choose any of the output primers.