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.
I'd like to try this but the install is difficult. Is there documentation?
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updated 3.6 years ago by
Ram
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written 13.3 years ago by
Lee Katz
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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.
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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updated 3.6 years ago by
Ram
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written 13.2 years ago by
lh3
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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.
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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updated 3.6 years ago by
Ram
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written 10.0 years ago by
lh3
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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).
Has anyone used this method? Does it work? http://biodoc.ist.unomaha.edu/wiki/In-silico_PCR#Methodology