Melting temp constraint removes all primers

[mhallerud]

I am running openPrimeR on a set of 65 sequences to develop primers for amplicon sequencing. I previously had the package up and running properly on a dummy dataset, but now that I'm running on my actual dataset I am having an issue. At Phase 2 a) in the design_primers function, I lose all of my primers at the melting_temp_range constraint. I have rerun design_primers with an extremely lax melting temperature constraint (0-100) to see if this was actually the issue, and I have also tried running on single sequences and a different set of sequences, but I am consistently getting 0 primers at this step. I also tried restarting R and reloading the package, but there's no change. I also checked my PATH variable in R and it's properly directing to the dependencies. I'm running R version 4.2.1 on Mac with MELTING 5.2.0, Oligo Array Aux 3.8.1. I'm a bit at a loss of what else to check, but I would really prefer using this package to running manually via Primer3. Help is greatly appreciated!

[Chessrein]

The same problem as mine. My codes are copied form tutorial. Please have a look. Thanks alot!

fasta.file <- system.file("extdata", "IMGT_data", "templates", 
                          "Homo_sapiens_IGH_functional_exon.fasta", package = "openPrimeR")
# Load the template sequences from 'fasta.file'
hdr.structure <- c("ACCESSION", "GROUP", "SPECIES", "FUNCTION")
seq.df.simple <- read_templates(fasta.file)
seq.df <- read_templates(fasta.file, hdr.structure, delim = "|", id.column = "GROUP")

l.fasta.file <- system.file("extdata", "IMGT_data", "templates", 
                            "Homo_sapiens_IGH_functional_leader.fasta", package = "openPrimeR")
template.df <- assign_binding_regions(seq.df, fw = l.fasta.file, rev = NULL)

settings.xml <- system.file("extdata", "settings", 
                            "A_Taq_PCR_design.xml", package = "openPrimeR")
settings <- read_settings(settings.xml)

optimal.primers <- design_primers(template.df[1:2,], mode.directionality = "fw",
                                  settings = settings)

[Chessrein]

But the same code can run in my pc's openprimer without the packages 'MELTING ', 'ViennaRNA ','OligoArrayAux ', 'MAFFT ' and 'Pandoc '. That is confuse me alot.

[tppf]

uninstall MELTING 5.2.0

[matdoering]

The package has been developed with MELTING 5.1.1. So, it may be that there are issues with newer versions. If you remove MELTING, the melting temperature calculations will default to the rule of thumb (less accurate than MELTING).

Can you check the values of the melting temperatures that are calculated when MELTING is installed? If the values are off that would explain why everything is filtered out.