In Silico Pcr Assay

Question

[Lee Katz]

Hi, does anyone have a method for in silico PCR? In other words, I have two primers and a genomic sequence and would like to have a prediction of a PCR result. A BioPerl solution would be perfect, but I won't snub my nose at other solutions. Thank you.

[Lee Katz]

Has anyone used this method? Does it work? http://biodoc.ist.unomaha.edu/wiki/In-silico_PCR#Methodology

Answer 1

[Casey Bergman]

Try Jim Kent's isPCR (which is the executable for UCSC's PCR mentioned by Pierre):

Source is here (zip file). Executables for different platforms are here.

[Lee Katz]

I'd like to try this but the install is difficult. Is there documentation?

[Casey Bergman]

Hi Lee. There is documentation in the README in this source directory, but as an alternative I've added a link to compiled executables. Inside the relevant directory you will find an isPcr.zip file which should run out of the box if you've chosen the right platform.

[Peter]

Easy to install if you are using conda, ispcr was packaged for bioconda: https://anaconda.org/bioconda/ispcr

[Lee Katz]

I am trying to install as a non-root user and I think it is assuming I am root.

Answer 2

[Pierre Lindenbaum]

Did you try:

• UCSC ePCR?
• NCBI ePCR
• or primer3?

[Pierre Lindenbaum]

There's a perl wrapper for primer3: http://www.bioperl.org/wiki/Module:Bio::Tools::Run::Primer3

[Lee Katz]

Primer3 seems to be command line and all-purpose. I'll try it out and see if it predicts my PCR product length. Thanks.

[Pierre Lindenbaum]

... but I think the two first would be better for a whole genome.

[Lee Katz]

I'm wondering what is best on the command line though, for high throughput.

Answer 3

[Wubin Qu]

Try:

http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/

or

http://biocompute.bmi.ac.cn/MFEprimer/

[Casey Bergman]

Thanks for posting this Wubin, it looks like a nice system that I wasn't aware of.

[Wubin Qu]

Please use the new server: http://www.mfeprimer.com/

Answer 4

[lh3]

I was directed here from seqanswers. Actually the best strategy is to use short-read mappers. Checking primer uniqueness is exactly the same as mapping Illumina paired-end reads. The short-read mappers are very fast and accurate. They also check mismatches and indels.

[Biomonika (Noolean)]

Yes, but then you need to figure out the orientation of the primers and their relative distance which is additional work.

[lh3]

I said "paired-end". Illumina paired-end reads have the same orientation as primers. The insert size of a read pair is exactly the same as the length of the PCR product. With a short-read mapper supporting paired-end mapping, you need to do little additional work while benefiting the speed, accuracy and sensitivity of mainstream short-read mappers which are usually more sophisticated.

[Peter]

It ought to work OK, but the task is not exactly the same: an in-silio PCR tool ought to be able to use heuristics for fuzzy matching which do no apply to short-read mappers (e.g. importance of mismatches at the 5' versus 3' end).

Answer 5

[Biomonika (Noolean)]

I am using command line version of primersearch. It outputs all amplicons and size of the products.