metagenomics pcr in triplicate-why?

[LLG]

Does anyone know why it is common to do the pcr for metagenomics in triplicate (same conditions and sample DNA) and then pool the products? Is it just to get enough DNA for the rest of protocol or is there some other reason?

[cmazzoni]

Hi,
I will give my explanation, I hope it matches other people's reason to do the PCR in triplicate. There several aspects and they all involve PCR-created biases.

As you have a collection of organisms, you don't expect every organisms' DNA to be amplified at the same cycle. Something that randomly starts to be amplified in the first cycles will be over-amplified in relation to other sequences. Other than that, DNA from less frequent organisms may be missed in one PCR because they are rare in the DNA fragments collection. Yet another reason is if you want to check for quantitative differences (which is always tricky). Because of the bias created by the exponential amplification, PCR-based sequencing can easily over/under-estimate the organisms frequency. The triplicate will buffer biases created in a single PCR. Having said all that, three is an arbitrary number of course, but it's the minimum number to help overcoming problems in one reaction.

Hope that helped.
Cheers.