Figure 2. Quantitative amplicon sequencing schematic. A A mixed-composition sample. Each circle represents a single pollen grain from four different species, in this example, indicated by the four colors. The numbers of each pollen grain in the mixed sample are shown below (i.e. mixed sample #1 had 11 grains from red, 7 grains from yellow, etc.). B This mixed sample is amplified via PCR across four different thermal cyclers, each with different cycle number “treatments”. C Each of these samples are then uniquely indexed to keep track of sample identify and PCR cycle treatment. All samples are pooled, post-PCR, and run on a single Illumina MiSeq lane, where they are sequenced. D After sequencing, samples are de-multiplexed. E Read abundance for each species in each sample is used to calculate the Ct value from a simplified read abundance ‘curve’. F These Ct values are used to calculate the proportion of each species in each sample (i.e. mixed sample #1 had more red pollen grains, a lower Ct, and subsequently a higher relative proportion in the sample).