A brief discussion of our process can be found here. As with any technique, there are concerns and it is important to consider some of the following when designing PCR.
Inherent to an amplicon approach for library preparation are a biases associated with primer choice and the biochemistry of PCR. For every pair of PCR primers (forward and reverse), there will be an inherent bias. If you are trying to target a specific organism that bias is a good thing, because it means your primers will preferentially amplify your organism of choice. However, in universal approaches (such as 16S sequencing), which are trying to screen multiple organisms, that bias is a negative. A number of researchers have published articles on the efficiencies of different primers. One of the more thorough ones is Klindworth et al. 2013 which provides suggestions for different length amplicons. However, sometimes the efficiency of the primer is less important that the ability to compare the results to other published studies. In this case, it is important to use the primers available from those studies or projects (for example the Earth Microbiome 16S project). We have a number of commonly used primers, in addition to less commonly used ones. If you have a specific primer you would like to use, we encourage you to check our assay list here and see if those primers are there. If you don’t see them, please feel free to email us the sequence to check (it is always a good idea to confirm the sequence of the primer we will be using as many primers with slight modifications have the same name). Even if we don’t have a specific primer set, it is not difficult for us to order them.
A second concern when determining which primer set to use is the thermal conditions for those primers. We use a standard profile for all our PCR’s, unless there is a specific request otherwise. If you have defined expectations, it is important to communicate them to us. We try to be as flexible as possible to our client’s needs, however we cannot take responsibility for project design or set up outside of our standard approach unless a written request is made to modify our protocol.
Generally, high quality DNA performs well and those samples move through our pipeline quickly. From some starting material it is difficult to get high quality DNA or enough DNA and PCR does not work as well. We do however offer multiple troubleshooting options, including amplifying with a more robust Taq, cleaning up the sample DNA to remove inhibitors, adding more or less DNA than our standard protocol, running at different annealing temperatures, increasing the number of cycles, or adding solutions to help increase PCR efficiency (BSA for example). These services are extra, unless we extract your sample, so be sure to check with our support team if you aren’t sure if your samples will need them.
Quantitative PCR (also known as RealTime PCR) is a common molecular biology approach using dye labeled probes (or SybrGreen in the mastermix).
Our primary use for this technique is quantifying the number of 16S copies in a sample (comparable to a plate count). Although this is a useful number, in a mixed sample it can be a little misleading as different organisms have different numbers of 16S in their genome. We do have other assays besides the 16S and can set up new assays if necessary. Contact our support group for more information.