added fasta parsing for compatible primers and revisited primer dimer evaluation

[jonas-fuchs]

This PR allows to parse a fasta file with primers that should not form dimers with the new scheme and will hopefully allow the design of multi-schemes. Moreover it applies more stringent primer dimer filtering. First, primer dimer standrad temp was set to a lower temp and then primer end overlaps are always checked for all primers not only qpcr. as the parsed fasta can increase the computation time dramatically for schemes with a lot of primers, multiprocessing was implemented.

[jonas-fuchs]

needs better explanation that now its a check for all permutations

[jonas-fuchs]

This now implements more stringent primer dimer considerations. Also the primer dimer solve at the end of the scheme is now more stringent. For each potential pool of alternate primers the primer cloud is first checked against all primers in the scheme that do not lead to primer dimers to inhibit that primer switching produces new primer dimers with existing primers. By that we only switch primers when it we are sure that new primer dimers can only occur from primer dimers in the primers we want to switch. As this is computationally more extensive for larger schemes, multiprocessing was implemented.

@wm75 If you have time to check this also, I would be super happy. In any case, because I changed some settings in the config, the Galaxy tool likely has to be slightly modified.

[jonas-fuchs]

and moreover added multi-processing at different steps of the respective workflows.

[Copilot]

Pull request overview

Adds support for filtering newly designed primers against a user-provided FASTA of “compatible primers”, tightens primer-dimer evaluation (Tm, ΔG, and end-overlap checks), and introduces multiprocessing to reduce runtime impact.

Changes:

• Add --compatible-primers FASTA input and filter primer/probe candidates against it.
• Replace direct calc_dimer().tm checks with a unified primers.is_dimer() (Tm/ΔG/end-overlap).
• Parallelize several expensive steps (primer/probe search, dimer resolution).

Reviewed changes

Copilot reviewed 8 out of 8 changed files in this pull request and generated 3 comments.

Show a summary per file

File	Description
varvamp/scripts/scheme.py	Uses primers.is_dimer() and parallelizes heterodimer resolution; returns richer dimer info.
varvamp/scripts/reporting.py	Changes dimer report output format and includes structure/ΔG.
varvamp/scripts/qpcr.py	Parallelizes probe discovery and probe→amplicon assessment; switches to primers.is_dimer().
varvamp/scripts/primers.py	Adds FASTA parsing for external primers, new dimer logic (is_dimer), and multiprocessing primer filtering.
varvamp/scripts/logging.py	Updates config validation for new params and extends goodbye messages.
varvamp/scripts/default_config.py	Introduces PRIMER_MAX_DIMER_DELTAG and moves END_OVERLAP into primer dimer constraints; lowers default max dimer Tm.
varvamp/command.py	Adds CLI arg, wires new multiprocessing signatures, integrates compatible-primer filtering and updated dimer solving output.
docs/how_varvamp_works.md	Documents new multiprocessing behavior.

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[Copilot]

Pull request overview

Copilot reviewed 8 out of 8 changed files in this pull request and generated 3 comments.

Comments suppressed due to low confidence (1)

varvamp/command.py:152

• In qpcr mode, --threshold is no longer required and now silently defaults to 0.9. This is a CLI behavior change that isn’t mentioned in the PR description and may surprise users/scripts that relied on the previous requirement. If intentional, please call it out in release notes/docs; otherwise consider restoring required=True.

    QPCR_parser.add_argument(
        "-t",
        "--threshold",
        metavar="0.9",
        type=float,
        default=0.9,
        help="consensus threshold (0-1) - higher values result in higher specificity at the expense of found primers"
    )

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[wm75]

Finally, regarding https://github.com/jonas-fuchs/varVAMP/pull/57/changes#r2732047859:

I guess you can write the primer dimer report as part of write_scheme_to_files instead of early in the process.
Specifically here:

varVAMP/varvamp/scripts/reporting.py

Lines 300 to 305 in 9c9f99f

	# write tsv file
	print(
	amp_name,
	amp_length,
	primer_name,
	primer[-1],

you have both the final (primer_name) and the original (primer[-1]) primer name available.
So, for each primer you're writing to that tsv, you could check based on its original name whether it's among the dimers, then write it to the dimer file under it's new name.

[jonas-fuchs]

@wm75 Thanks a lot for your review. If you find the time to double check after the implementations, it would be great!

[wm75]

@wm75 Thanks a lot for your review. If you find the time to double check after the implementations, it would be great!

on it.

[wm75]

For consistency, I'd change this to:

    # Create an iterator over the first n amplicons for processing
    # Amplicons are sorted first on whether offset targets were predicted for them,
    # then by penalty. This ensures that amplicons with offset targets are always considered last.
    amplicons = itertools.islice(
        sorted(qpcr_schemes_candidates, key=lambda x: (x.get("offset_targets", False), x["penalty"])),
        n_to_test
    )
    batch_size = int(n_to_test/n_processes)
    callable_f = functools.partial(
        process_single_amplicon_deltaG,
        majority_consensus=majority_consensus
    )

    # Create a pool of processes to handle the concurrent processing
    with multiprocessing.Pool(processes=n_processes) as pool:
        # process amplicons concurrently
        results = pool.map(callable_f, amplicons, batch_size)

[wm75]

All code changes look good and consistent to me.

I've tested various command line runs and all seems good.

What I didn't check is whether all changes are properly documented.

[wm75]

Suggested change

	from Bio.Seq import Seq
	from Bio.Seq import MutableSeq

[wm75]

Suggested change

	return str(MutableSeq(seq).reverse_complement(inplace=True))

just a simple micro-optimization that occurred to me while going through the code. Makes only a few seconds runtime difference overall.

[jonas-fuchs]

I'll carefully recheck documentation. Thanks alot for the review :)

All code changes look good and consistent to me.

I've tested various command line runs and all seems good.

What I didn't check is whether all changes are properly documented.