Leray et al 2013's CO1 primers are commonly used in labs looking to detect vertebrates. I've been trying to include them in my in silico prep work but they're so highly degenerate, I can't get regular bioinformatics tools to work with them.
The more common combination is (replacing the Leray R primer with Geller's R primer, see Table 1 in the above paper)
>mICOlintF
GGWACWGGWTGAACWGTWTAYCCYCC
>jgHCO2198
TAIACYTCIGGRTGICCRAARAAYCA
1) When I use blastn with and without -task blastn-short, I get no hits in my mitogenomes. Just empty output.
2) web-blast thinks the Ws are amino acids and says I should use blastp. Replacing all degenerate bases by N does work, but no results.
3) CRABS insilico_pcr does not find hits with the default error rate; if I increase it to 10%, I get suspiciously long PCR products that seem to be partial CO1s, partially other stuff.
4) isPcr from https://genome-test.gi.ucsc.edu/\~kent/exe/linux/ segfaults
5) De-MetaST-BLAST does... something? It seems to remote-blast with nr and that takes forever
6) EMBOSS primersearch finds nothing
7) https://genome.ucsc.edu/cgi-bin/hgPcr finds nothing
Is there a tool that can work with these?
Edit: I've now settled on running CRABS with a non-standard error rate of 10% (not the default 4.5%), followed by a length-filtering step. That seems to catch most CO1 products, removing <1% of the predicted products. Is there a better way?
