Genotyping by sequencing for estimating relative abundances of diatom taxa in mock communities

Abstract

Background: Diatoms are present in all waters and are highly sensitive to pollution gradients. Therefore, they are ideal bioindicators for water quality assessment. Current indices used in these applications are based on identifying diatom species and counting their abundances using traditional light microscopy. Several molecular techniques have been developed to help automate different steps of this process, but obtaining reliable estimates of diatom community composition and species abundance remains challenging.

Results: Here, we evaluated a recently developed quantification method based on Genotyping by Sequencing (GBS) for the first time in diatoms to estimate the relative abundances within a species complex. For this purpose, a reference database comprised of thousands of genomic DNA clusters was generated from cultures of Nitzschia palea. The sequencing reads from calibration and mock samples were mapped against this database for parallel quantification. We sequenced 25 mock diatom communities containing up to five taxa per sample in different abundances. Taxon abundances in these communities were also quantified by a diatom expert using manual counting of cells on light microscopic slides. The relative abundances of strains across mock samples were over- or under-estimated by the manual counting method, and a majority of mock samples had stronger correlations using GBS. Moreover, one previously recognized putative hybrid had the largest number of false positive detections demonstrating the limitation of the manual counting method when morphologically similar and/or phylogenetically close taxa are analyzed.

Conclusions: Our results suggest that GBS is a reliable method to estimate the relative abundances of the N. palea taxa analyzed in this study and outperformed traditional light microscopy in terms of accuracy. GBS provides increased taxonomic resolution compared to currently available quantitative molecular approaches, and it is more scalable in the number of species that can be analyzed in a single run. Hence, this is a significant step forward in developing automated, high-throughput molecular methods specifically designed for the quantification of [diatom] communities for freshwater quality assessments.

Keywords: Diatoms; GBS; Mock communities; Quantification; Relative abundance; Water quality assessment.

Fig. 1

Regression lines (blue and red) and coefficients of determination for relative abundance estimates per Nitzschia palea strain across mock samples. 95% confidence intervals (grey) are drawn around the regression lines

Fig. 2

Composition and relative abundance estimates of mock Nitzschia palea samples obtained by using Genotyping by Sequencing (GBS) and Light Microscopy (LM) methods. Correlation coefficients were calculated using the Pearson correlation test. The green text indicates a strong correlation (> 0.7), the orange text indicates a moderate correlation (0.5–0.7), and the red text indicates a weak or no correlation (< 0.5)