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

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..., Genomic DNA was extracted manually from all samples using a modified CTAB extraction procedure. The GBS protocol and sequencing followed Wagemaker et al. (2020) with minor modifications. In brief, extracted genomic DNA from the 36 samples was digested with two restriction enzymes (PacI and NsiI), and two indexed adapters were ligated to the digested DNA fragments. Each adapter incorporated a three-base pair unique molecular identifier (UMI) region to identify PCR duplicates within each library. The libraries were pooled and aliquoted in three portions to further prevent PCR bias. These aliquots were purified using QIAquick (QIAGEN), size selected for >150 bp fragments using AMPure XP beads (Beckman Coulter), and nick repaired using DNA polymerase I to repair nicks and improve PCR efficiency. The cleaned libraries were amplified (16 PCR cycles) using KAPA HiFi HotStart ReadyMix (Roche). The PCR reactions were combined, concentrated using QIAquick, size selected again for >150 bp fr..., Interactive Genome Viewer for bam and fasta files.
Excel for csv files., # Genotyping by Sequencing for estimating relative abundances of diatom taxa in mock communities

[https://doi.org/10.5061/dryad.gqnk98srr ](https://doi.org/10.5061/dryad.gqnk98srr)

## Description of the data and file structure

We acquired six *N. palea* strains from Thonon Culture Collection, France (TCC) [[53](https://bmcecolevol.biomedcentral.com/articles/10.1186/s12862-023-02104-2#ref-CR53 \"Rimet F, Chardon C, Lainé L, Bouchez A, Domaizon I, Guillard J et al. Thonon Culture Collection -TCC- a freshwater microalgae collection; 2018.\")] (Table [5](https://bmcecolevol.biomedcentral.com/articles/10.1186/s12862-023-02104-2#Tab5)). The cultures were grown in WC medium [[54](https://bmcecolevol.biomedcentral.com/articles/10.1186/s12862-023-02104-2#ref-CR54 \"Guillard RRL, Lorenzen CJ. Yellow-green algae with chlorophyllide c. J Phycol. 1972;8(1):10–4.\")] at 19 °C and on a 16 h light/8 h dark cycle. We routinely examined the live cultures under a Zeiss Axio Imager M2 microscope and transfe...