Fig 1.
Schematic illustration of study design.
Differently colored primer and library indexes represent unique index sequences used to identify the sample origin of reads generated after sequencing on an Illumina MiSeq. A total of 25 indexed primer sets were used, and the single and double PCR treatments were sequenced in separate sequencing runs comprised of three and two uniquely indexed libraries, respectively.
Fig 2.
Dissimilarity among results from indexed primer sets used on the same environmental sample.
Mean pairwise Bray-Curtis dissimilarity among sequencing replicates within and among indexed primer sets used to amplify each environmental sample (N = 11) using both a single (left) and double (right) PCR protocol. Bray-Curtis dissimilarity value of 0 indicates two samples are exactly identical while a value of 1 indicates they are exactly different. For the single PCR treatment, the mean Bray-Curtis dissimilarity values for within-primer index comparisons (M = 0.027, SD = 0.0050) were significantly lower than those of among-primer index comparisons (M = 0.685, SD = 0.196; Welch’s t(10.01) = 11.13, p < 0.00001).
Fig 3.
Effect of indexed primers on the proportional abundance of a representative OTU.
Proportional sequence counts for a representative OTU (annotated to the gastropod genus Elysia) from each of 11 independent environmental samples (distinguished on the horizontal axis and delineated by vertical grey dashed bars). Each sample was amplified using two indexed primer sets (represented by closed or open points), each of which were sequenced in multiple replicates (single PCR N = 3; double PCR N = 2). Within a sample, variance among instances of the same symbol (among filled points or among closed points) represents variance across the three different sequencing libraries using the same indexed primer set. Variance between symbols (between open and closed points) indicates variance in relative abundance due to differences between primer indexes.