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WASP (a Web-based Allele Specific Primer designing tool) is an application that designs allele-specific (AS) primers for detecting SNPs and mutations. WASP input can be either key searches (Section A) or SNPs with flanking sequences (Section B). Users can input key searches (SNP IDs or Gene Names), consequently, WASP returns the search result as a graphical interface including SNP information, AS primers (if exist) and PCR condition. SNPs with flanking sequences can be input to the system via the text box or the file uploading module. Additionally, users are able to adjust some primer picking parameters based on the desired PCR condition (Section C).

The system architecture can be described by the workflow diagram


Section A: Search for AS primers of SNPs present in the SNP Database

WASP enables users to design AS primers for SNPs in the local SNP database including dbSNP, Hapmap, and JSNP. Users whom wish to genotype a set of interested SNPs can simply skip the SNP sequence preparation step. They only have to identify a key search (SNP ID and Gene Name) and a specific database. Key searches can be entered manually in the query box (See Figure I).

Example

SNP ID: 1500

Gene Name:
CYP2D6

Figure I WASP input interface

According to the database described above, the database name need to be selected for a gene name query(See Figure II), in contrast to SNP ID query that already points to a specific database itself.

Figure II Database selection. dbSNP (blue oval), HapMap (red oval) and JSNP (green oval) with number of SNPs in CYP2D6 gene is shown.

After a database is selected, a selectable SNP list for AS primer design is shown, therefore, users can select a set of SNP from this table for designing AS primers (Figure III). Then click the design button.

Figure III Selectable SNPs in CYP2D6 gene of dbSNP database.


Section B: User-defined Input

WASP is designed to handle user-defined input in the standard FASTA format. The input can be entered manually or uploaded (Figure IV). Generally, 5' and 3' flanking sequence length should be at least 40 bases each. Upper and lower case is insignificant. Other letters, including numbers, spaces are neglected. SNP location can be defined by bracketing [wild type allele/ mutant allele] and IUPAC ambiguity code.

Example
Bracket
>SNP_ID
...CCCATCTACCTTTTCGAGTTGAAGTTATTTTGAGAACACATCCCCTAAG[A/T]GTTTATCTTGGTTATTTGC
AATAAAACATTTTTCATTTGAAAATTACCCACAGTTATTATCC...
IUPAC ambiguity code
>SNP_ID
...CCCATCTACCTTTTCGAGTTGAAGTTATTTTGAGAACACATCCCCTAAGWGTTTATCTTGGTTATTTGCCT
AACTAATAAAACATTTTTCATTTGAAAATTACCCACAGTTATTATCC...

Make sure that the SNPs location are defined in the same way throughout the input sequences. If a bracket is chosen, it must be applied to all sequences. See an example input file here.

Figure IV User-defined input. a) file uploading and b) text box filling.

The Penultimate Primer Position Mismatching

To improve reaction specificity, it is recommended to introduce additional mismatches at the penultimate base of the AS primers. Due to different mismatches have different destabilizing effects, it is essential to consider terminal and penultimate mismatch together. For example, if the terminal mismatch is “weak”, it is likely that a “strong” additional mismatch should be introduced. The base change follows Table I proposed by Little, 1995. It presents the selection of penultimate base. Each entry in the table presents a choice of the underlying mismatch base at the penultimate location. For example, if the 3'-end terminal base has its mismatch as A---G, and the base next to this terminal appears to be C, then the base to be inserted in AS primer should be "A''. This will guarantee that the PCR hybridization will stop when it encounters another mismatch at the penultimate location. Users can select this feature by checking the check box.



Table I Mismatching reference table


 

Section C: Primer3 Parameter Adjustment

Primer Size (integer)
Optimum, minimum, and maximum size of a primer oligo. Primer3 will likely to pick up primers with the close length to Optimum size. Minimum size basically has to be greater than 0 and less than or equal to maximum size (0<min<max), which is no greater than 36 . This limit is set by maximun oligo size for which Primer3's melting-temperature is acceptable. The deflault Optimum, minimum and maximum size are 20, 18 and 36 respectively.
GC% (float)
Optimum, minimum, and maximum allowable percentage of Gs and Cs in any primer. Optimum GC% influence primer selection close to this number. The deflault Optimum, minimum and maximum GC% are 50, 20 and 85 respectively.
Tm(float)
Optimum, Minimum, and Maximum melting temperatures (Celsius) for a primer oligo. Primer3 will not pick oligos with temperatures smaller than Min or larger than Max, and with default conditions will try to pick primers with melting temperatures close to Optimum. Primer3 uses the oligo melting temperature formula given in Rychlik, Spencer and Rhoads, Nucleic Acids Research, vol 18, num 21, pp 6409-6412 and Breslauer, Frank, Bloeker and Marky, Proc. Natl. Acad. Sci. USA, vol 83, pp 3746-3750. Please refer to the former paper for background discussion. The deflault Optimum, minimum and maximum Tm are 55, 40 and 65 respectively.
Max Tm
Diff (float)
Maximum acceptable difference between the melting temperatures of the left and the right primers. The default value is 100.
Max Self Complementarity (decimal)
The maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between left and right primers. Local self-complementarity is taken to predict the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. The scoring system gives 1.00 for complementary bases, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed.The default value is 8.00.
Max 3' Self Complementarity (decimal)
The maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored global alignment score when testing for complementarity between left and right primers. The 3'-anchored global alignment score is taken to predict the likelihood of PCR-priming primer-dimers. The default value is 3.00.

Max polyX in primer (integer)

The maximum allowable length of a mononucleotide repeat, for example AAAAAA. The default value is 3.
Number primer pairs to return
Number of allele specific primers to return.
In-silico PCR filtering

Search for all possible PCR product(s) in the genome using BLAT (Kent 2002; Genome Res., 12:656-664) in order to improve primer pair specificity. To select this feature, check the in-silico PCR attribute and specify the maximum product size. The default value is 4000 bases.

Mispriming filtering
Check if primers appear in a nucleotide sequence library of a sequence avoid amplifying, for example repetitive sequences, or possibly the sequence of gene family that should not be amplified.


Output

Results provide a sumary of AS primers and their relative information. Target SNPs/mutations and the resulting AS primers will be visualized in graphical objects. When moving a mouse over each object, there will be a popup window whose contents show related primer conditions. If no AS primer is reported, only input sequence and original primer parameter conditions will be presented (Figure V).

SNP ID
SNP identity
Input Sequence
Input Sequence from 5' to 3' dirrection, identifying SNP by braketing wild-type allele and mutant allele
Sequence Size
Input sequence length
Primer Picking Parameters
The summary of parameters that are used by WasP to design AS-primers
Pos.

Primer position starting from 5' end of the input sequence

Len

Primer length

Tm, GC, Self Any, Self End
Tm, GC, Self Complementarity and Self 3' Complementarity of a primer

Figure V SVG graphical view of selected SNPs shown by blue dots on the SNPs. Table indicates if the selected SNPs have their AS primers successfully designed. By clicking on the checked row in the table, the SVG view of designed AS primers will be displayed. Top 5 oligo primers are displayed for each design where green and red arrows show wild and mutant AS primers respectively. The blue arrow indicates the common primer. Sizes of the primers are displayed above the arrows. Upon mouse-over action on each primer, the detailed information will be presented in the box next to it.

If WASP can not find any AS primers...

WASP adopted primer3 core engine to do the design task. In case that no AS primer is found, some primer3 parameters should be relaxed e.g. increasing self-complementarity parameters and and varying max and min oligo melting temperatures. For example, for an A-T-rich region around a SNP you might have to decrease minimum melting temperature.

 

Citation

If you found WASP was helpful, please cite:

Wangkumhang P, Chaichoompu K, Ngamphiw C, Ruangrit U, Chanprasert J, Assawamakin A, Tongsima S: WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations. BMC Genomics 2007, 8:275 [http://bioinfo.biotec.or.th/WASP] [Abstract] [Full Text] [PDF]

 

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Copyright (c) 2006-2007, National Center for Genetic Engineering and Biotechnology. All rights reserved.

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