PrimalScheme

web interface

Server running primalscheme v1.2.1

  • FASTA containing one or more reference genomes.
  • Min/max will be set at 5% either side of target.
  • A short name/prefix for your scheme, no spaces.

View demo inputs

Job status:

Region: -

Primers considered: 0

0%

Created by Josh Quick and Andy Smith. Artwork by Oli Pybus.

About

primalscheme is a tool for designing primer panels for multiplex PCR. It uses a greedy algorithm to find primers for tiling amplicon generation for multiple reference genomes. It works best on viral isolates of known lineages e.g. outbreak strains.

Instructions

  1. Download complete genomes from GenBank and concatenate into a single FASTA file.
    • e.g. cat "sequence (1).fasta" "sequence (2).fasta" > input.fasta
    • primalscheme does not currently support inputs with ambiguity codes.
  2. Align all sequences using Clustal Omega.
  3. The genomes should be complete and roughly the same length.
    • Primers are only designed within the limits of the alignment.
    • The first reference will be used for the coordinate system in the output scheme.
  4. Check the 'Result Summary' tab then the 'Percent Identity Matrix' link to check the identity matrix of all sequences.
    • Check that the maximum sequence divergence is not more than 5%.
    • If your sequences are more divergent, it may be necessary to separate them into multiple independent schemes; the 'Phylogenetic Tree' tab may be useful for this.
  5. Remove sequences with 99-100% identity to other genomes in the file as these will bias the primer selection
  6. Run primalscheme on your FASTA file.
    • Optionally, set amplicon length as required by your sequencing technology.

Protocol

We strongly recommend using the PCR conditions outlined in the Nature Protocols paper.

Command Line Interface

If you prefer a CLI, primalscheme is available as a python package. See our GitHub repo for further details.

Citing

If you use primalscheme please cite:

Quick J et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc. 2017 Jun;12(6):1261-1276.