The Whitehead program must be set up and on the path in order for eprimer3 to find and run it.
Primer3 picks primers for PCR reactions, considering as criteria:
All of these criteria are user-specifiable as constraints.
eprimer3 can also pick hybridisation oligos that are internal to the product.
Techniques for avoiding problems include a thorough understanding of possible vector contaminants and cloning artifacts coupled with database searches using blast, fasta, or other similarity searching program to screen for vector contaminants and possible repeats. Repbase (J. Jurka, A.F.A. Smit, C. Pethiyagoda, and others, 1995-1996, ftp://ncbi.nlm.nih.gov/repository/repbase) is an excellent source of repeat sequences and pointers to the literature. eprimer3 now allows you to screen candidate oligos against a Mispriming Library (or a Mishyb Library in the case of internal oligos).
Sequence quality can be controlled by manual trace viewing and quality clipping or automatic quality clipping programs. Low- quality bases should be changed to N's or can be made part of Excluded Regions. The beginning of a sequencing read is often problematic because of primer peaks, and the end of the read often contains many low-quality or even meaningless called bases. Therefore when picking primers from single-pass sequence it is often best to use the INCLUDED_REGION parameter to ensure that eprimer3 chooses primers in the high quality region of the read.
In addition, eprimer3 takes as input a Sequence Quality list for use with those base calling programs
(e.g. Phred, Bass/Grace, Trout) that output this information.
Try setting the '-explain' option.
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If the '-explain' flag has been used, as in the example, then statistics are output describing the number of primers that were considered and rejected for various reasons.
Headers describing the input file name and the names of the output columns are displayed.
The best 5 primer pairs are displayed with the product size.
The reverse sequence is displayed as the reverse complement to the input forward sense sequence.
The 'Start' positions are given counting from the start of the sequence for both the forward and reverse primer (and for the internal oligos).
Note that if you compare the results to the output from the public Primer3 web site you will see a difference in the reverse primer positions - this is because the original Primer3 program reports the reverse primer positions as counted from the 3' end. The convention in EMBOSS is to report both forward and reverse features as counted from the 5' end, so the reverse primer positions are given counted from the 5' start of the sequence.
The Whitehead Institute program that is run by this program is available from:
http://primer3.sourceforge.net/
(Then see the link Download and then 'Release 1.1.4')
Earlier versions are also supported. We expect later versions to also work - please contact the EMBOSS team if you find any problems.
The Whitehead Institute's primer3 program states:
We request but do not require that use of this software be cited in
publications as:
Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386
Source code available at http://fokker.wi.mit.edu/primer3/. The paper above is available at http://jura.wi.mit.edu/rozen/papers/rozen-and-skaletsky-2000-primer3.pdf