Primer Tester

About

This tool tests degenerate primers against a sequence database (typically a genome).

Loads primers from a fasta file (primers can contain IUPAC nucleotide codes).
Uses NAF-formatted file as database (NAF on GitHub).
Scans both strands of the database sequences.
Supports up to 2 mismatches (per primer).
Finds amplicons delimited by any two primers.
Tested on Linux and Windows

Download

primer-tester-0.5.0.zip (August 27, 2020) - shared under the zlib/libpng license.

Usage

primer-tester.pl [OPTIONS] >result.txt

Options:

--query FILE.fa - Specifies query file (mandatory).

--db DB.naf - Specifies database file (mandatory).

--max-n-mismatches N - Allow up to N mismatches when matching each primer, from 0 to 2 (default = 0).

--max-amplicon-size N - Maximum length of amplified sequence, including both primers (default = 1000).

--max-n-primer-hits N - Use at most N locations of individual primers (1000000 by default).

--max-n-amplicon-hits N - Report at most N amplicons (1000 by default).

Dependencies

Perl
Sort::Versions perl module (installed using, e.g., sudo cpan Sort::Versions)
unnaf (from NAF tools)
ripgrep

Example:

Input query file ("1.fa"):

>S-D-bact-0008-c-S20 AGRGT TYGAT YMTGG CTCAG >S-D-bact-1391-a-A-17 GACGG GCGGT GWGTR CA

Command:

primer-tester.pl --query 1.fa --max-n-mismatches 2 --max-amplicon-size 10000 --db GCA_000001405.28_GRCh38.p13_genomic.naf >1-result.txt

Output file ("1-result.txt"):

CM000685.2 Homo sapiens chromosome X, GRCh38 reference primary assembly 71665003 4520 S-D-bact-1391-a-A-17 S-D-bact-1391-a-A-17 GGCGGGCAGTGAGTACA TGTACTCACCGCCCGTC

Output format

Each line starting from non-space is a sequence name. Following lines are hits found in this sequence.

Each hit has these fields (separated by spaces): start coordinate (1-based), amplicon size, name of the 1-st primer, name of the 2-nd primer, sequence matching 1-st primer, sequence matching 2-nd primer.