Regardless of sequencing platform, bioinformatic analyses can be daunting.

Ecological quality ratio (EQR) scores were calculated according to the Dutch standards for both morphological and DNA-based taxon lists, using the QBWat software version 5.33 [44] (with redundancies removed as described previously). Research that does cover a broader variety of WFD monitoring samples often deals with differences in taxonomic resolution between morphological and DNA analyses [16,25]. While beginners may be intimidated by the complexity of bioinformatic analyses, we recommend that non‐specialists start with these resources to analyse their data.

Each entry on the combined lists was classed into one of the following categories: (1) “found”, where there was an exact match between both lists; (2) “identified at a different level”, when there was a match, but either one of the lists had a higher-level identification; (3), “putative misidentification”, in cases where two different species from the same genus were listed on the respective lists; (4) “missing in reference” when the morphologically identified species was not covered by the DNA reference database; (5) “not found”, when the taxon was covered in the reference database, but only encountered in the morphological list; (6) “extra”, when the taxon was only encountered in the DNA list. Temperature is a key factor for DNA preservation. Initial denaturation was performed at 98°C for 30 seconds, followed by 30 cycles at 98°C for 5 seconds, 50°C for 5 seconds and 72°C for 15 seconds, followed by final elongation at 72°C for 5 minutes. In 18 samples, more taxa were found with DNA than with morphology, in 26 samples more taxa were obtained with morphology. Use the link below to share a full-text version of this article with your friends and colleagues. These include studying the responses of terrestrial biodiversity to land‐use change (Wood et al., 2017; Schmidt et al., 2018), monitoring ecological dynamics of lake macroinvertebrates (Bista et al., 2018), and biodiversity assessment in marine arthropods (Fonseca et al., 2010; Sinniger et al., 2016).

Freshwater macroinvertebrate samples were collected in the Hoogheemraadschap Rijnland monitoring district in 2010 and 2012 by ecological survey company Aquon (Leiden, the Netherlands). Nuclear mitochondrial pseudogenes can be identified based on in‐frame stop codons, insertions or deletions, but are otherwise challenging to detect (Song et al., 2008).

With rarefaction the sorted samples still had 2.22 times as many MOTUs and 1.40 times as many taxa; neither was significantly lower than without rarefaction.

Now that the use of DNA barcoding for the identification of species [1] has proven its merit, research is shifting towards the integration of molecular identifications in ecological and biodiversity assessments across different biomes [2–5].

PLoS ONE 14(12): the mitochondrial cytochrome c oxidase I (COI) gene is the standard DNA barcode for identifying animal species. For 13 out of 24 separately processed mollusc samples (one sample did not include molluscs) we were unable to amplify molluscs using the standard approach for DNA extraction and PCR. First round PCRs were performed in 20 μl reactions containing 1x Phire Green Reaction Buffer, 10 μg BSA (Promega, Madison, WI, US), 0.5 mM dNTPs, 0.4 μl Phire Hot Start II DNA Polymerase (Thermo Fisher, Waltham, MA, US), 0.65 μM of each primer and 2.0 μl of template DNA. Concerns about the limited taxonomic and geographical coverage of sequence databases for DNA metabarcoding studies are regularly raised. Allows end‐users to follow a default analytical parameter settings or configure a specific pipeline.

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