PrimalScheme v3.0.2

primer panels for multiplex PCR

Essentials

Lowercase letters, numbers, and hyphens only. Your input will be automatically formatted to meet these requirements.

Min/max length will be ±10%.

How many pools should the scheme use?


Fine tuning

What frequency should a mutation be to be included in primer design?

Default is 10% amplicon size. Provide a value to override.

Getting Started

Input files

  • You must provide a fasta file (max 10 MB) for each target, up to a maximum of 10 files per scheme.
  • You can provide aligned or unaligned sequences:
    • Aligned inputs will be used directly.
    • Unaligned inputs will be aligned using MAFFT.
  • You should select genomes that are representative of the diversity of the target.

Running PrimalScheme

  1. Create a job and specify your required parameters using the form above:

    • Amplicon length: Smaller amplicons will be more sensitive but also require more primers.
    • Number of pools: A minimum two pools are required for overlapping amplicon schemes.
    • Minimum base frequency: Bases below this frequency in the fasta will be ignored in primer design.
    • Target overlap: Larger overlap increases the redundancy but increases the number of amplicons.
    • Backtrack: allow the algorithm to try alternative paths to prevent gaps (increases run time).
    • High-GC: increases the min/max primer GC content for high GC genomes.
  2. Read and accept the terms and conditions (first visit).

  3. Upload your fasta file(s).

  4. Your job will be queued, and running status displayed live.

  5. Results will be available for download, and a link will be emailed to you.

Primary output files

Files required for ordering primers and analysis.

  • primer.bed: A file containing the coordinates and sequence of the primers.
  • reference.fasta: The primary reference genome used for the scheme (first genome in file).

Advanced Usage

Input files

If there are too many genomes, Treemmer can be used to phylogenetically downsample, increasing the representation of minor clades.

For files larger than 10MB it is required to run primalscheme locally. Instructions can be found on the primalscheme GitHub repo.

Additional output files

Supporting files that may be be useful for analysis/debugging.

  • amplicon.bed: A file containing the coordinates of the amplicons.
  • primertrim.amplicon.bed: A file containing the coordinates of the amplicons after primer trimming.
  • plot.html: An interactive plot of the scheme.
  • primer.html: An interactive plot of the primers.
  • {reference}.png: Files containing the interactive scheme plot for each reference genome.
  • config.json: A file containing the detailed run parameters.
  • file.log: Log file for debugging.

Using the scheme

Ordering Primers

Primers can be ordered from a variety of suppliers. We developed bed2idt to convert the primer.bed file into an Excel plate ordering template for IDT. Oligos are reorganised into different plates for each pool to make pooling easier. On the IDT website select the product '25 nmole DNA Plate Oligo' in 96 well format. Upload the Excel template and import the plates. Select Plate Type 'PCR', Normalization type 'Normalized Yield' and Quantity(nmol) '10' and then apply settings to all plates.

Publishing Schemes

If the scheme is useful please consider uploading it to labs.primalscheme and make it visible to the community. Instructions on how to do this can be found on the primal-page GitHub repository.

Protocols

Primers are designed for PCR conditions described in; nCoV-2019 sequencing protocol v3 (LoCost)