I isolated DNA from a mouse fecal pellet from the large intestine using the QIAamp DNA Stool Mini Kit and sequenced using Illumina Miseq targeting the V4 region. We readily detected enterobacteriaceae using specific primers and qPCR but upon sequencing there was very little detection of enterobacteriaceae. We should really expect to see plenty of enterobacteriaceae because the treatment we are using (DSS) has been shown to increase these populations. Would using a different database help in this situation? We are using Silva.
Thanks
How may E. coli do you expect? Recall that in a normal community, E. coli might be quite rare (<1%). Are you sure that your primers would amplify E. coli? You might try classifying your sequenes with the RDP files, but I doubt it will make much of a difference.
Pat
I think the E. coli should be somewhere between 1-5% after a DSS treatment. We tried primers that were specific for E. coli as opposed to the Enterobacteriaceae but with these E. coli primers there seemed to be mininmal expression. I’m wondering whether we might have low E. coli/Enterobacteriaceae since they more commonly associate with the mucosal layer of the intestine.