Assessing dinophyte biodiversity in Bavarian lakes (Germany) by 18S v4 amplicon sequencing

Seventeen surface plankton tow samples were collected from piers at thirteen localities in Upper Bavaria (Germany) in April 2017 using a plankton net (mesh size 20µm). The localities included eleven lakes (one lake was sampled at two sites) and one subsidiary river, to cover standing and flowing bodies of water as well. Four sites had been sampled twice. Environmental DNA was extracted using the Genomic DNA from Soil kit (Machery-Nagel; Düren, Germany) following the manufacturer's protocol. The small subunit (SSU or 18S) of the ribosomal RNA (rRNA) operon V4 region (~410 bp) was the amplification target. Due to PCR biases or PCR errors that may artificially increase diversity, each PCR reaction was performed in triplicates. Forward and reverse primers were those used by Xiao et al. (2017) (DOI: 10.1007/s12010-016-2358-3). DNA amplification (PCR) for subsequent Illumina amplicon sequencing (Illumina; San Diego, USA-CA) was carried out using 5ng/µl template DNA, 1 µM of each primer and 2x KAPA Hifi HotStart Ready Mix (Roche; Penzberg, Germany). Resulting PCR products were visualised in 1% agarose gels and were purified using AMPure XP Beads (Beckman Coulter; Brea, USA-CA). Dual indices and Illumina sequence adapters were attached by means of an Index PCR using the Nextera XT Index Kit (Illumina), and final PCR products were again purified using AMPure XP Beads. The library was validated using an Agilent 2100 Bioanalyzer Software and a DNA 1000 Chip (Agilent Technologies; Santa Clara, USA-CA) to verify the size of the resulting fragments. The final DNA libraries were equimolarly pooled and run in a MiSeq System (Illumina) after combining the denatured PhiX control library (15%) and the denatured amplicon library. Some 6.5 million 2 x 300 bp paired-end reads were produced and demultiplexed into seventeen samples from thirteen sites. Using Trimmomatic (v0.38), 3'-ends of the reads were trimmed based on read quality information. PEAR (v0.9.10) with default settings was used to merge the paired-end reads. Sequences, which could not be merged, were discarded. Primer-matching sequence segments were truncated from the amplicons by cutadapt (v1.9) and amplicons were only kept in the sequence pool, if both, the segment of the forward and of the reverse primer could be found. Remaining sequences were filtered for further quality features by vsearch (v2.3.0). Sequences were discarded, if they were outside a 50 bp radius above or below the median length of the primer-truncated amplicon (~387 bp), if they carry any ambiguity or if the expected number of miscalled bases of a sequence (sum of all base error probabilities of a sequence) was above 1. Chimera were predicted also by vsearch utilising the UCHIME algorithm with default settings in de-novo mode for each sample separately and removed from the sample files. About 4 million sequences passed all filtering steps and were used as input for the OTU-clustering, which was done by the tool Swarm (v2.1.8) with default settings. The most abundant amplicon of each OTU-cluster was used as an OTU representative. These sequences were annotated by the RDP classifier implemented in mothur (v1.38.1) using the Ref_NR99 version of release 128 of the SILVA SSU sequence set using a reference with a confidence cutoff of 90. The annotation of each representative sequence was used as annotation of the OTU cluster as well and added to the corresponding line of the OTU table.