Hi everyone.

Just looking for some thoughts on this. My thinking is that I shouldn't use touchdown PCR to amplify 16S regions in this case because it will cause bias. The primers will already bind better to those sequences that are a perfect match with them (and as we know, "universal" primers are not quite so universal). So, by using touchdown PCR, I'd essentially be amplifying the issue as the whole point is to reduce non-specific binding. Am I right in thinking this?

My problem is, I have already done it. I had a batch of 160 samples sequenced last year and only afterwards did I realise that I should have used a standard PCR. The issue now is that I have my second batch of 320 samples ready to PCR. They are from the same experiment and I want to compare the microbiomes between them and the previous batch. Therefore, I think I should keep the methods identical. Should I use the touchdown PCR again just to keep it consistent with the previous batch??

Many thanks for any input!