In Silico Pcr Assay
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10.8 years ago
Lee Katz ★ 3.1k

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.

pcr bioperl • 18k views
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Has anyone used this method? Does it work? http://biodoc.ist.unomaha.edu/wiki/In-silico_PCR#Methodology

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10.8 years ago

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.

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

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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.

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

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

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10.8 years ago

Did you try:

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

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

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

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10.8 years ago
Wubin Qu ▴ 160
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Thanks for posting this Wubin, it looks like a nice system that I wasn't aware of.

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

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10.8 years ago
lh3 33k

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.

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

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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.

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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).

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7.6 years ago

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

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