Degenerate primers | undefined columns selected

[thierryjanssens]

Hi,

I am trying to check the specificity of the following degenerate primer pair from the literature.
Unfortunately, it is not producing any results.

BLAST alignment processing, refreshing in 20 seconds...
BLAST alignment processing, refreshing in 20 seconds...
BLAST alignment completed in 71 seconds
Error in [.data.frame(hits, location_columns) :
undefined columns selected

The method give a specificity overviewwith another candidate primer pair and the examples from the vignette work as well.

Kind regards,

Thierry

[MVesuviusC]

Hi Thierry, what primers are you testing so I can try to replicate the error? It may be that your primers just don't match anything in the database.

[thierryjanssens]

Hello Matt,

thanks for reaching out.
I am sorry, I thought it was in my post.

This is the code I used. The primers are from literature and producing good results in the qPCR.

library(primerTree)
dsr1 = search_primer_pair(name='drs1', 'ACSCACTGGAAGCACGGCGG', 'GTGGMRCCTGCAKRTTGG', total_primer_specificity_mismatch=19, num_permutations=32)

pdf("dsr1.pdf")
plot(dsr1, ranks=c("class"))
dev.off()

thanks,

T.

[thierryjanssens]

The same error is returned when using the following primers:

'AGAGTTTGATCMTGGC', 'TACCTTGTTACGACTT'

[MVesuviusC]

I tried running both of these primer sets directly through primerblast (filling in the ambiguous bases by hand) and got zero hits, which is why you're getting that error message. If these primers are working in the lab, they clearly they're amplifying something, but it may be worthwhile to find a sequence they're supposed to amplify and make sure there isn't some issue with the primer sequence compared to the known sequence.

[thierryjanssens]

That is strange.
In the reference organism the dsrA primer sets has binding sites on two positions in the genome:

[MVesuviusC]

I'm not sure, if you take the green and yellow sequences and try them in primer blast, do they find hits?