Hello all,
I am having a problem with one question from a reviewer. We have metabarcoded a sample for which we know the species we should be able to retrieve. There are a number of species that were not identified through metabarcoding and we class them as false negatives. The reviewer asks if we are sure that our primer sets would recover those species from the sample and suggests a way to test it would be to make sure the primers work on the already known barcodes.
This is probably incredibly basic but I am not sure how to do it. My only guess was to blast the primer set and try to manually find the species but I am sure there is a better way (besides, by blasting just a primer, I get no results anyway). Thanks a lot for your help!
I'd do two approaches.
1. Use BLAST to see if the primers are predicted to create a product in those species.
2. Set up PCR reactions using those barcodes in samples that you KNOW has those species.
Sometimes primers don't amplify everything they should - smaller products amplify more often than larger, rare DNA might get "missed" compared to common DNA, etc.
Or the issue might be with the DNA extraction if it's more difficult to get good quality DNA out of some organisms compared to other (e.g. Gram positive vs. Gram negative bacteria).
Good luck!
Daniel Souto-Vilarós did you previously test the primers on DNA from the species you want to identify on the sample?
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