Development of genetic phytoplankton monitoring

Abstract

Unicellular microorganisms are the most abundant and diverse lifeforms, being the basis for the existence of other organisms. However, microscopic analysis of microorganisms is a slow process, and it is often impossible for the tiny creatures. To uncover microbial diversity via molecular tools, two library preparation techniques for high-throughput sequencing (HTS) of ribosomal RNA (rRNA) genes and rRNA were developed using phytoplankton as a model target group. Bioinformatics pipelines for data trimming and operational taxonomic unit (OTU) clustering were incorporated into the study. At first, a cost- and time-efficient workflow for fluent barcoding and size trimming of sequencing templates was established. By targeting the sequencing to the same region of the template molecule, data could be optimally used for OTU-based algorithms. The lack of an extensive validation of molecular tools for phytoplankton diversity analysis gave reason to compare DNA and RNA preservation, extraction and HTS data trimming methods. All processes impacted the HTS results of a mock community, with known biomasses and carbon content for each species. In the rRNA gene-based community profiling, species richness was accurately revealed, but the relative abundances of species were biased due to the high species-specific variation in the rRNA gene copy numbers. However, rRNA-based analysis reflected the relative biomasses more closely and was recommended for studying the diversity of eukaryotes. Even in the RNA based analysis, PCR amplification with gene-specific primers can bias the outcomes, as real universal primers for amplifying eukaryotic and prokaryotic ribosomal genes are not available. Therefore, a primer-independent rRNA workflow was developed to allow directional 5′-end HTS. A significant advantage of this method is that it allows simultaneous sequencing of all domains of life. The new workflow was applied to analyse water samples from 83 Finnish lakes. Results uncovered an incredibly high diversity of organisms and partly agreed with, and partly complemented, phytoplankton microscopy results.