Hi all
We use a low annealing temperature to try and capture as much diversity as possible using "universal" V4 - V5 primers. Given that we are trying to capture sequences which do not exactly match the primer, I run my downstream analysis allowing effectively unlimited primer mismatches. My theory is that if the sequence is not 16S at all, it will a) be relatively low abundance, b) won't properly align and be discarded and c) won't be classified in taxonomy assignment.
I'm concious that this is a controversial approach, and wondered if anyone had any thoughts / uses a similar approach?
We use a low annealing temperature to try and capture as much diversity as possible using "universal" V4 - V5 primers. Given that we are trying to capture sequences which do not exactly match the primer, I run my downstream analysis allowing effectively unlimited primer mismatches. My theory is that if the sequence is not 16S at all, it will a) be relatively low abundance, b) won't properly align and be discarded and c) won't be classified in taxonomy assignment.
I'm concious that this is a controversial approach, and wondered if anyone had any thoughts / uses a similar approach?