A note on TaqMAN Probe Sensitivity
Our method of quantitative amplicon sequencing (below) applies the general theory of quantitative PCR. Before introducing this method, a natural question regarding our approach is why we did not use fluorescence-based quantitative PCR, such as TaqMan probes (Thermo Fischer Scientific, Waltham MA, USA). We note here that we did develop and test TaqMan probes for the small region that distinguished the fourClarkia species within the manufacturers recommended design specifications. However, these probes, with 1-4 species-specific SNPs, were not sensitive to fluoresce exclusively enough in the target species, and therefore did not allow us to distinguish among any of the four Clarkia species. Thus, the TaqMan chemistry was not sensitive enough to generate reliable relative abundance information. We are unaware of other published reports discussing this sensitivity, which was also not clear to the manufacturer, and thus raise this point here for researchers interested in applying fluorescent probes to quantify relative abundance using a small number of SNPs.