Testing the effect of pollen exine rupture on metabarcoding with Illumina sequencing

Pollen metabarcoding has received much attention recently for its potential to increase taxonomic resolution of the identifications of pollen grains necessary for various public health, ecological and environmental inquiry. However, methodologies implemented are widely varied across studies confounding comparisons and casting uncertainty on the reliability of results. In this study, we investigated part of the methodology, the effects of level of exine rupture and lysis incubation time, on the performance of DNA extraction and Illumina sequencing. We examined 15 species of plants from 12 families with pollen that varies in size, shape, and aperture number to evaluate effort necessary for exine rupture. Then created mock communities of 14 of the species from DNA extractions at 4 levels of exine rupture (0, 33, 67, and 100%) and two levels of increased lysis incubation time without exine rupture (2 or 24 hours). Quantities of these DNA extractions displayed a positive correlation between increased rupture and DNA yield, however increasing time of lysis incubation was associated with decreased DNA yield. Illumina sequencing was performed with these artificial community treatments with three common plant DNA barcode regions (rbcL, ITS1, ITS2) with two different primer pairings for ITS2 and rbcL. We found decreased performance in treatments with 0% or 100% exine rupture compared to 33% and 67% rupture, based on deviation from expected proportions and species retrieval, and increased lysis incubation was found to be detrimental to results.