PriSM

Primer Selection and Matching for Amplification of Viral Genomes


  *This tool will find valid primer candidates from input genomes and pair the primers to create amplicon candidates.

Primer & Amplicon Selection Conditions:

Primer size (bp) Maximum number of nucleotides in primer  Default: 20
Primer overlap length (bp) Number of bases primers can overlap by in design phase  Default: 19
Minimum GC percentage Minimum GC content of each primer  Default: 45
Maximum GC percentage Maximum GC content of each primer  Default: 55
Minimum Tm (°C) Lower limit of primer melting temperatures  Default: 56
Maximum Tm (°C) Upper limit of primer melting temperatures  Default: 65
Maximum number of degenerate bases allowed (bp) Maximum number of IUPAC bases allowed in final primer  Default: 3
Maximum number of monomer bases allowed (bp) Number of bases in a mononucleotide run within a primer  Default: 4
Primer concentration (nM) Concentration at which primer will be synthesized (used for Tm calculation)  Default: 50
Salt concentration (mM) Salt concentration in primer stock (used for Tm calculation)  Default: 50
Valid base minimum frequency The minimum frequency of a given base at a given position in the alignment at which the base is considered valid  Default: 0.1
Valid degenerate maximum frequency The upper limit of conservation in an alignment at a given position, above which the base is called for the majority at that position. Below this, a dengenerate base is called.  Default: 0.85
Maximum self-complement bases allowed within a single primer (bp) Number of bases within a primer that are self-complimentary  Default: 10
Valid gap minimum frequency The frequency of genomes within an alignment that must contain a gap at a given position for that gap to be represented in the consensus  Default: 0.8
    
Primer blast minimum hit frequency Minimum frequency of hits for a given primer back to the genomes in the alignment  Default: 0.8
Maximum amplicon length (bp) Upper limit for amplified product length  Default: 900
Minimum amplicon length (bp) Lower limit for amplified product length  Default: 500
Amplicon overlap length (bp) Desired overlap between adjacent amplicons in the panel  Default: 100
Maximum degenerate primer combination Maximum number of unique primer combinations as determined by the permutations of degenerate bases within a primer   Default: 8
Maximum complement bases in primer pair Number of bases that can be complimentary between a forward and reverse primer in a pair  Default: 15
    
Genome name (Single word without space or symbol[-, _, #, ...], e.g. HIV) Single word (no spaces or symbols) to indicate template. This title will appear in primer name.  

Upload genome multi-fasta file (Required)
(With extension .fasta only. No spaces No symbols in filename):
File used to make alignment, with extension .fasta.  

After you click "Submit", the results will be sent to you via email
Your Email Address



Copyright © Special Project Group, Broad Institute of MIT and Harvard     Last Modified 2009-06-23