Biomonitoring and surveillance with short- and long-read metabarcoding

Abstract

The aim of this project was to develop applied metabarcoding methods to aid both the monitoring of environmental water quality and surveillance of airborne fungal phytopathogens. An Illumina short-read metabarcoding method was developed which is now in active use by the UK Environment Agency to determine the abundance of diatom species, feeding into the classification of water bodies for the EU Water Framework Directive. Further work was undertaken to future proof this method by comparing the diatom assemblages of three English rivers as determined by light microscopy, the developed Illumina short-read metabarcoding method and long-read nanopore metabarcoding. The river and method comparison study showed that the light microscopy was the outlier and potentially largest source of error as the two separate metabarcoding methods performed very similarly. A study was undertaken to compare the airborne fungal communities in six locations in eastern England over a one month period in 2015 to assess metabarcoding as a potential surveillance tool for the introduction of phytopathogens and the utility of the current UK pollen network for sample acquisition. This study highlighted issues with contamination at stages within the metabarcoding laboratory preparation protocols which made the bioinformatics analysis problematic; however, recommendations are made for procedures to reduce contamination in metabarcoding studies. The final study characterised a week's data from the eastern England fungal spore samples with a novel long PCR amplifying the entire ribosomal tandem repeat - including the intergenic spacer - to investigate the regions full utility in the light of nanopore sequencing.