Hi, I am studying microbial communities at cold seeps site near methane emission spots on the seafloor. Using 16S, we are able to amplify sequences, some samples up to 3% of the sequences were associated in SILVA database to methanotrophs. However, detection remains absent of methanotrophs if I use primers targetting pmoA-pmoC 374F / pmoA 344R. Now I assumed that the amount of pmoA sequences may be too low, but just in case has anyone encountered similar situation in marine samples?
I haven't used these primers but could you try to run an assay (PCR OR qPCR) with mcrA primers to see if you detect any methanogens. This might give you an insight about what could have gone wrong in your assay with pmoA. Although, primers are different for different community, it might suggest you if it is the result of very low number of methanotroph or any artefact due to the functional target.
Its surely not the answer for your question, but hope you find the solution soon.
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