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

Abstract

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.

Plant biodiversity assessment through pollen DNA metabarcoding in Natura 2000 habitats (Italian Alps)

“…Other studies have applied capture enrichment which led to more accurate results avoiding PCR biases 40 . In addition, pollen structure can lead to over and under-representation of some taxa as demonstrated by previous studies 41 . Studies investigating predefined mock communities have attempted to give insight to quantitative issues 42 – 44 , but more controlled experiments with detailed knowledge of the eDNA ecology and simulations in all ecosystems would be needed to correlate the observed with the true abundance 42 .…”

Pollen DNA metabarcoding and related methods in global change ecology: prospects, challenges, and progress

“…There are off-the-shelf kits that work well, along with cheaper extraction methods based on phenol and chloroform; acetone-based DNA isolation has also been effective (Gouker, Guo, & Pooler, 2020). Some studies have shown that it is possible to extract and sequence DNA from pollen without a lysing step (Swenson & Gemeinholzer, 2021). The high-throughput capabilities of DNA metabarcoding are reliant on the addition of sample-specific nucleotides which allow the identification of hundreds of individual samples following pooling into a single sample sent for sequencing (multiplexing).…”

“…These standards can then be applied when designing a project and when assessing papers during peer-review. Some methods for the various steps involved in pollen DNA metabarcoding have been compared (Swenson & Gemeinholzer, 2021;Tommasi, Ferrari, Labra, Galimberti, & Biella, 2021), however, there remains a lot of work to be done before any general recommendations can be made.…”

Using DNA Metabarcoding to Identify Floral Visitation by Pollinators

“…Numerous DNA isolation methods exist which can influence the quality of the DNA template [124,125]. Membrane-based isolation techniques are most commonly used for pollen metabarcoding studies, providing a fast and simple way of yielding DNA, although are costly [124].…”