Sample-size and bioinformatics independent, consistent patterns among
diatom metabarcoding data from lake sediments
Abstract
Diatoms (Bacillariophyceae) are widely used as bioindicators of present
and past water quality because they inhabit the vast majority of aquatic
ecosystems, are very diverse, highly sensitive to a variety of
environmental conditions, and are characterized by silicified cell walls
that favor their long-term preservation in sediments. Alongside with
traditional morphological analyses, metabarcoding has become a valuable
tool to study the community structures of various organisms, including
diatoms. Here, we aimed to test whether the quantity of sediment sample
used for DNA extraction is affecting the results obtained from
high-throughput sequencing (metabarcoding) of the diatom rbcL region by
isolating DNA from 10 g and 0.5 g (wet weight) of lake surface sediment
samples. Because bioinformatics processing of metabarcoding data may
affect the outcome, we also tested the consistency of the results from
three different pipelines. Additionally, the agreement between
metabarcoding data and morphological inventories of corresponding
samples were compared. Our results demonstrate highly uniform patterns
between the diatom rbcL amplicons from 10 g and 0.5 g of DNA extracts
(HTS 10 and HTS 0.5, respectively). Furthermore, metabarcoding results
were highly consistent among the data sets produced by different
bioinformatics pipelines. Comparing results from metabarcoding and
microscopy, we identified some taxonomic mismatches, which are related
to the common issue of incompleteness of the sequence databases, but
also to inconsistencies in diatom taxonomy in general and potential
dissolution effects of diatom valves caused by high alkalinity of the
investigated lake waters. Nevertheless, multivariate community analysis
demonstrated highly similar results between data sets identified by
microscopy and metabarcoding, further confirming that metabarcoding is a
viable alternative for identifying diatom-environment relationships.