In silico PCR primer match sequences

[blakem...@gmail.com]

Hello, 

I am using Metacoder to perform in silico PCR and have a good set of primers that amplify my gene of interest, but it requires me to allow 2 mismatches. I am now looking to identify the specific bases that are different in the primer binding sites so I understand any potential issues here and can identify needed base degeneracy. Is there a way to pull out the primer binding site sequences from the references using Metacoder? 

Thanks!

Blake

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A slightly edited version of the code is: 

seqs <- ape::read.FASTA(fasta_path)

cat(names(seqs)[1])

data <- extract_taxonomy(seqs, regex = "^(.*)\ (.*)",

                         key = c(id = "obs_info", "class"),

                         class_sep = ";")

data

pcr <- primersearch(data, 

                    forward = c("F" = FORWARDSEQ), 

                    reverse = c("R" = "REVERSESEQ), 

                    mismatch = 20)

Tax <- taxon_data(pcr)

[Zachary Foster]

Hi Blake,

I think that is doable. I have done it before for my own research a while ago. I am currently getting ready to release a big update to metacoder and I need to go through the in silico PCR code, so I will look into that today or tomorrow and get back to you. I think I will be able to put the primer binding sites in the output for the new version.

You mention that it requires you to allow for 2 mismatches; where are you seeing that? Does setting the ` mismatch` option to `0` work? Thanks,

-Zach

[Blake Hanson]

Hi Zach,

Sounds great! I think this would be an excellent addition to metacoder. 

For the 2 mismatch detail, I meant the mismatch = 20 parameter. Due to the lengths of my primers I know that the 20% mismatch translates to 2 bases. 

Thanks!

Blake

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Blake Hanson

E-mail - blakem...@gmail.com

[Zachary Foster]

Hello Blake,

Sorry for the delay! It was harder than I expected and the holidays slowed things down for a while, but I think I have a solution for you. I suggest you use the dev version on github (soon to be the CRAN version):

devtools::install_github("ropensci/taxa")
devtools::install_github("grunwaldlab/metacoder")

and look at the example in ?primersearch.
There has been a lot of changes in this last update and the online documentation will need to be updated, but the function help is up to date.
Let me know if you have questions.

Best, 

Zach