Best primer design software

Question

[4galaxy77]

hi,

I'm designing some PCR primers for some microsatellite regions - in total probably more than 50. I've been using primer3, but found it to be very laborious and difficult, as there isn't an option to exclude repeat regions (there is an option to remove mononucleotide repeats, but not, say GAGAGA). Plus I'm not to keen on the GUI pieces of software.

Has anyone got any recommendations for programs which say, accept a text file full of sequences and designs primers for them, rather than having to input them all individually, but with a high degree of control over the design specs. Possibly some kind of R plugin or Linux command line tool?

Thanks

[Benn]

When I saw the title of your question my first thought was: Primer3. But after reading the details, you want something else than primer3...

I think primer3 is the most successful designer available, and it is even available on linux

http://primer3.sourceforge.net/primer3_manual.htm#installLinux

What does your ideal primer designer tool look like? I mean how should it handle repeats?

[4galaxy77]

Ah OK, well maybe I will have a mess around with it in Linux, thanks. Essentially, I have to exclude polynucleotide repeats from the binding sites, so for example, I can't include GATGATGAT. primer3 allows you to exclude strings of mononucleotide repeats (GGGG), but I'm not aware of it doing this for polynucleotides. This is kind of trivial if you only need to design a couple of primers, but when you have 50-100, it can be very time consuming to scan through a 300bp sequence.

Also, it would be useful to be able to have a large txt file with all of my sequences in, and submit them in batch rather than individually, but perhaps the Unix version of primer3 does this. I'll have a look.

Thanks!

[Benn]

I have to admit that I haven't designed any primers for ages (must be ~5 years ago), so I might not be the best teacher here...

That being said...

If you look at the online designer http://primer3.ut.ee/ you can see the option for excluded regions. The second option enables you to mark the regions not to include for primer design. You can mark the beginning and end of your repeats there. Just a thought...

[mks002]

You can take a look at this Batchprimer3. Hope you find the link useful.

[Charles Plessy]

there isn't an option to exclude repeat regions

You can provide a mispriming library.

[WouterDeCoster]

You can mask the fasta files for repetitive sequences (NNNNN....) to make sure no primers will be selected in those.

[4galaxy77]

Yeah, thanks, that's what I've been doing but I was more hoping there would be something which automatically didn't select repetitive sequences, rather than having to manually exclude them. You can automatically exclude GGGGG, for instance, but not GAGAGA

[WouterDeCoster]

We typically mask all sequences in the repeatmasker track (UCSC genome browser). You can do this quite automatically, but this might not cover all GAGAGA sites. A custom script could do that...

Also don't forget to mask common SNPs.

[friveroll]

For low complexity regions you can use RepeatMasker http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker, it is also available to install on linux, bur you need to request academic license for some dependencies.

The output is your sequence with .....NNNNNN...... in the lox complexity regions of your sequence

Answer 1

[Charles Plessy]

The EMBOSS software suite has a wrapper for primer3, that you may find easier to use.

Answer 2

[h.mon]

There are several pipelines for microsatellite primer design, and most (if not all) of them wraps primer3 along some repeat recognition and retrieval, passing target/exclude regions to primer3 to avoid designing primers over the repeats. To name a few: MISA, QDD, FullSSR.