jonas-fuchs / varvamp

Design degenerated primers on highly variable alignments for full genome sequencing or qPCR. Specifically developed for viruses.

variable VirusAMPlicons (varVAMP) is a tool to design primers for highly diverse viruses. The input is an alignment of your viral (full-genome) sequences.

varVAMP

For a lot of virus genera it is difficult to design pan-specific primers. varVAMP solves this by introducing ambiguous characters into primers and minimizes mismatches at the 3' end. Primers might not work for some sequences of your input alignment but should recognize the large majority.

varVAMP comes in three different flavors:

SINGLE: varVAMP searches for the very best primers and reports back non-overlapping amplicons which can be used for PCR-based screening approaches.

TILED: varVAMP uses a graph based approach to design overlapping amplicons that tile the entire viral genome. This designs amplicons that are suitable for Oxford Nanopore or Illumina based full-genome sequencing.

QPCR: varVAMP searches for small amplicons with an optimized internal probe (TaqMan). It minimizes temperature differences between the primers and checks for amplicon secondary structures.

Documentation

• Installation
• Preparing the data
• Usage
• Output
• Wet-lab protocol
• How it works
• FAQ

Already established primer schemes

We, in collaboration with specialists for the respective viruses, have already designed and wet-lab evaluated primer schemes for various viral pathogens. All the input data and varVAMP outputs are freely available here.

If you design primers for a particular pathogen, we will be happy to include them in this repo and make it freely available. Just contribute via an issue or pull request!

Citing varVAMP (coming soon)

Please cite with the respective DOI of the version you used:

varVAMP: automated pan-specific primer design for tiled full genome sequencing and qPCR of highly diverse viral pathogens.

(paper currently under preparation)

---

Important disclaimer: For the primer design, varVAMP uses primer3 to check if digested kmers of a sequence are potential primers. Some of the functions for this were adapted from primalscheme and I do not claim credit.

The remaing code is under the GPLv3 licence. The code is WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.

varvamp's Issues

threshold for conserved nucleotides

Can you explain it in more detail?
-t 0.89, --threshold 0.89 threshold for conserved nucleotides

Which of the following is more similar in meaning?

1. 89% nucleotides in probe and primers are conserved in all genomes;

2. or, >89% nucleotides other than primers and probes are conserved in amplicon;
3. or, >89% of all genomes maybe be amplified

END_OVERLAP config setting not getting logged

config.END_OVERLAP is a setting that does not get echoed to the log file.
I'm also wondering whether it got placed into the qPCR section of settings in the default config by accident because the code appears to use it for regular primers, too.

Error response from daemon

$ docker pull quay.io/biocontainers/varvamp
Using default tag: latest
Error response from daemon: manifest for quay.io/biocontainers/varvamp:latest not found: manifest unknown: manifest unknown

could you tell me How fix it?

Warning about deprecated Biopython functionality

With recent enough versions of Biopython varVAMP triggers the deprecation warning from:

https://github.com/biopython/biopython/blob/4fc5158970b7970907fb932eae15eb779fdea839/Bio/Application/__init__.py#L40-L49

varVAMP should do as suggested in the warning and call blast directly via stdlib subprocess.

Problem combining qpcr mode with primer blast

when trying to combine mode qpcr with -db I'm running into:

Traceback (most recent call last):
  File "/home/wolma/miniconda3/envs/varvamp/bin/varvamp", line 10, in <module>
    sys.exit(main())
             ^^^^^^
  File "/home/wolma/miniconda3/envs/varvamp/lib/python3.11/site-packages/varvamp/command.py", line 554, in main
    probe_regions, final_schemes = qpcr_workflow(
                                   ^^^^^^^^^^^^^^
  File "/home/wolma/miniconda3/envs/varvamp/lib/python3.11/site-packages/varvamp/command.py", line 450, in qpcr_workflow
    query_path = blast.create_BLAST_query_qpcr(qpcr_scheme_candidates, data_dir)
                 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
  File "/home/wolma/miniconda3/envs/varvamp/lib/python3.11/site-packages/varvamp/scripts/blast.py", line 62, in create_BLAST_query_qpcr
    name = f"{primer_type}_{qpcr_scheme_candidates[amp][primer_type][1]}_{qpcr_scheme_candidates[amp][primer_type][2]}"

KeyError: 'probe'

which, at first glance, looks like a rather generic error that wouldn't depend on exact inputs, but let me know if you need more details to reproduce this.

Combination of qPCR and BLAST db fails

  File "/usr/local/tools/_conda/envs/mulled-v1-88ec11123572a51b245ba097dc0b273940132dc7e25597f386c2d705a3f21960/bin/varvamp", line 10, in <module>
    sys.exit(main())
  File "/usr/local/tools/_conda/envs/mulled-v1-88ec11123572a51b245ba097dc0b273940132dc7e25597f386c2d705a3f21960/lib/python3.10/site-packages/varvamp/command.py", line 557, in main
    probe_regions, final_schemes = qpcr_workflow(
  File "/usr/local/tools/_conda/envs/mulled-v1-88ec11123572a51b245ba097dc0b273940132dc7e25597f386c2d705a3f21960/lib/python3.10/site-packages/varvamp/command.py", line 455, in qpcr_workflow
    amplicons, off_target_amplicons = blast.primer_blast(
  File "/usr/local/tools/_conda/envs/mulled-v1-88ec11123572a51b245ba097dc0b273940132dc7e25597f386c2d705a3f21960/lib/python3.10/site-packages/varvamp/scripts/blast.py", line 240, in primer_blast
    off_target_amplicons, amplicons = predict_non_specific_amplicons(
  File "/usr/local/tools/_conda/envs/mulled-v1-88ec11123572a51b245ba097dc0b273940132dc7e25597f386c2d705a3f21960/lib/python3.10/site-packages/varvamp/scripts/blast.py", line 206, in predict_non_specific_amplicons
    amplicons[off_target]["penalty"][0] = amplicons[off_target]["penalty"][0] + config.BLAST_PENALTY
TypeError: 'float' object is not subscriptable```