Development and testing of an 18S rRNA phylogenetic microarray for marine sediments

Abstract

There is increasing interest in finding new, more efficient methods for routine monitoring of anthropogenic effects on benthic biodiversity and ecosystems. A range of molecular methods have been developed for assessing biodiversity the last decades. Particularly interesting are microarrays targeting phylogenetic marker genes, such as the small subunit of ribosomal RNA in eukaryotes (18S rRNA). This method can detect a large number of taxonomic groups in several samples simultaneously within a relatively short time and has the potential for incorporation in automated remote sensing pipelines. In this study we developed and tested a microarray for eukaryotes in marine sediments. The probes were designed to target 18S rRNA OTUs obtained through metabarcoding of marine sediments. The resulting microarray was tested using both a spiked sample consisting of 50 plasmid-clones and further, samples of genomic DNA extracted from marine sediments. We developed a filtration pipeline to eliminate noise and reduce the number of false positives, making it possible to detect and quantify most of the OTUs with ≥ 0.1% abundance in the spiked sample. Our data indicated that the microarray was specific at higher taxonomic levels. However, the specificity decreased with increasing sequence similarity suggesting cross-hybridization between closely related OTUs. When using genomic DNA isolated from marine sediment there was a positive correlation between hybridization intensity signals and abundance of sequencing reads, suggesting a quantitative behavior of the microarray. Overall, the data suggest a potential for microarrays as a tool for high throughput sediment monitoring.

Keywords: Benthic; Biodiversity; Ecosystem; Environment; Genomic DNA; Microarray; Monitoring; Probe.