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).