Issues on running independently

[544728460]

If I have already clustered the unaligned nucleotide sequences manually and only want to design primers for these clustered fasta files seperately, which script should i use? And if i have already got some primers of of these clustered fasta files and only want to evaluate the interaction of these primers to acquire a primer set without conflicts, how can i run the specific scripts?

[joybio]

@544728460 You can refer to the process in the multiPrime.py script, starting from Rule 7, to design primers for each cluster in parallel using the command multiPrime-core.py. If you want to continue generating a primer set, you can proceed with the later steps in the process. In Rule 17, the tools mfeprimer-3.2.6 and finDimer.py are used to check primer compatibility, and you can use them separately. Additionally, if you have a small number of clusters, you can use the multiPrime2 program (https://github.com/joybio/multiPrime2-GUI), which directly accepts clustered fasta files as input. It offers two algorithms (multiPrime1 and multiPrime2) that can be used. However, it cannot generate primer sets by itself.

[544728460]

Thanks Junbo! I have found the script 'multiPrime-core_V20.py' and there are still some unclear tips on this script:
For input paramaters:
1)"--plen" limits the length of primers. But can the primer length only be a fixed number not a suggested range? For example, 18 to 25?
2) What's the difference between the 'Max number of degenerate' and 'Max degeneracy of primer'?
3)For "--entropy", what are the boundary values of this parameters? Minimum or Maximum values?
4)How to understand "--fraction" or "match fraction"? Does it mean the specific primer desigend can only align to a fraction of the input sequence not all？
5）If i want to analyse the primers' specificity, which script should i use? Also is it available for users to build a local database for primer specificity analysis on their own?

For the output files of script 'multiPrime-core_V20.py':
These column names seem still unclear for me :'Position', 'Entropy of cover (bit)', 'Entropy of total (bit)', 'nonsense_primer_number', 'Optimal_coverage', 'Mis-F-coverage', 'Mis-R-coverage', 'Information'. Would you mind elaborating them further?

Thanks so much!