Abundance of airborne pollen for nine grass species, measured by qPCR, UK, 2016-2017

The qPCR data was collected using a QuantStudio 6 Flex Real-Time qPCR machine (ThermoFisher Scientific). Species-specific primers were designed to target the ITS2 region of abundant grass species in the UK including Anthoxanthum odoratum (sweet vernal-grass), Arrhenatherum elatius (false oat-grass), Cynosurus cristatus (crested dog's-tail), Dactylis glomerata (cock's-foot), Lolium perenne (perennial ryegrass), Phleum pratense (Timothy), Poa pratensis (smooth meadow-grass), grass species within the genera Alopecurus/Agrostis, and one probe that was found to be degenerate and unable to discriminate grass species.

Of 1,400 daily aerial samples, 1,210 were selected for downstream molecular analysis. Samples were excluded if pollen could not be reliably extracted due to large volumes of rainwater in collection tubes.

Quantitative PCR runs with PCR efficiencies less than 85% and greater than 115% were not used for further analysis (efficiency of qPCR data used in downstream analysis ranged between 88.5% and 106%). Data points with a large standard deviation between three technical replicates (>6.95, based on the upper quartile range of the data) were removed. In addition, samples which amplified before 10 cycles and after 38 cycles were removed to reduce the chance of detecting false positive or false negative amplification respectively. The reliability of the data was evaluated based on the positive and negative controls.