Error when running design_primers()

[shm0wt]

Hi,

I came across this while trying to design primers for ONT amplicon sequencing, and it seems pretty ideal for what I want - except that when I run the actual primer design, I get an error! Unsure if I'm making a mistake with my inputs or there is some sort of bug happening here?

My code:

library(openPrimeR)

targets = read_templates(targets_file) #read fasta sequences including 3kb flanking regions
targets = assign_binding_regions(targets, fw = c(1,2999), rev = c(1,2999)) #define flanking regions

settings.xml <- system.file("extdata", "settings", 
                    "A_Taq_PCR_design.xml", package = "openPrimeR")
settings <- read_settings(settings.xml) #may need some tweaks?

my_primers = design_primers(targets[1:10], settings = settings)

This produces the following output from R:

#####
# (FW) Primer design for forward primers
#####
# Phase 1: Initialization of primers
# Phase 2: Selection of primers
a) Filtering of primers
	o primer_length: 3990 primers remaining (100%)
	o gc_clamp: 1507 primers remaining (37.77%)
	o gc_ratio: 835 primers remaining (20.93%)
	o no_runs: 798 primers remaining (20%)
	o no_repeats: 798 primers remaining (20%)
	o melting_temp_range: 0 primers remaining (0%)
b) Relaxation of constraints
Checking melting temperature set coverage:
Melting temperature set @ NA with 0 primers (0 coverage)
	o Augmenting primer coverage for 3990 of 3990 target primers.
Basic Coverage: on_target
Basic Coverage: off_target
Computing constrained coverage ...
Error in seq.default(min(annealing.temp), max(annealing.temp), 2) : 
  'from' must be a finite number
In addition: There were 50 or more warnings (use warnings() to see the first 50)

[mattheiter]

In the filtering output of the log you can see how your constraints impact the amount of primers available after each filtering step. It seems that after the melting temperature filter, there are no more primers remaining which leads to an error. Can you review your design constraints and adjust to make the melting temperature range wider?

[shm0wt]

I have played around with the design constraints a bit, and it doesn't seem that this helps unfortunately. Even setting an arbitrarily large range doesn't make a difference e.g.

constraints(settings)$melting_temp_range = c("min" = 0, "max" = 1000)

Still filters out all the remaining primers at the end of the a) filtering_of_primers step.

I guess this melting temp analysis relies on the 3rd party MELTING tool? In which case, it may be that something is going wrong with that software rather than the OpenPrimeR() package itself. I haven't previously used MELTING and installed it for use with OpenPrimeR(). It runs from the command line and OpenPrimeR() does not throw a warning message when the library is loaded, but perhaps it is still not working as intended?

[mattheiter]

Ah, maybe I misinterpreted the log and it's already at the duplicate filtering step. Please try adjusting the constraints there.