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Raw cutadapt miseq output - minibarcode 18S-V7 of macroalgae

Citation

Ørberg, Sarah (2021), Raw cutadapt miseq output - minibarcode 18S-V7 of macroalgae, Dryad, Dataset, https://doi.org/10.5061/dryad.1jwstqjw9

Abstract

Macroalgae are key primary producers in North Atlantic and Arctic coastal ecosystems, and tracing their fate and distribution is vital to improve our understanding of their ecological role and provision of ecosystem services. Recent advances from environmental DNA (eDNA) have added a new capacity to fingerprint and trace macroalgae. However, further development of resources for amplifying and identifying macroalgal eDNA are much needed. Here, we examined the performance in terms of resolution and specificity of two 18S primers (18S-V7 & 18S-V9) recently applied in identifying macroalgae from eDNA. We also built a local barcode database for primer 18S-V7 with 31 widespread Arctic and North Atlantic macroalgal species to complement the existing DNA databases. Furthermore, we applied metabarcoding of eDNA to identify macroalgae in Arctic marine sediments (Disko Bay, W. Greenland) and evaluated the contributions from our local barcode database. We identified macroalgal DNA from 19 families across 11 orders in surface (0-1 cm, with both primers) and sub-surface (5-10 cm, with 18S-V7 primer) sediments. The barcode database developed here with the 18S-V7 primer improved the identification of unique families, from 16 to 19 families, thereby strengthening the taxonomic assignment possible relative to pre-existing barcode reference sequences. Overall, this study demonstrates the feasibility of eDNA to resolve contributions of macroalgae in Arctic marine sediments, and enhances the fingerprinting resolution. We thereby document a novel pathway to answer key questions on the ecological role and fate of macroalgae in the Arctic.

Methods

To develop the barcode database, macroalgal samples were collected along the coast of Denmark and Southwest and East Greenland in connection with various research expeditions (Table 1). The samples include 44 individuals (37 species) of red, green, and brown algae.

Macroalgal tissue was homogenized using liquid nitrogen and grinded with a mortar and pestle, which were sterilized between samples with 10% bleach and 70% ethanol. DNA from 100 mg of grinded sample was extracted with the Nucleospin Plant II kit (Macherey-Nagel) following the manufacturers protocol.

We targeted the 18S rDNA using the 18S-V7 primer pair (Table 3). PCR reactions contained 5 μl of QIAGEN Multiplex PCR Master Mix, 1 μl (1.0 μM) of each primer, 1 μl of DNA template, and 2 μl of PCR water. The PCR protocol for 18S-V7 was followed as detailed in table 3. A PCR blank with DNAfree water and a mock sample, with a mix of algal DNA samples in different DNA concentrations (Appendix B, table B1) was included with the samples. PCR amplicons were prepared for sequencing with QIAquick PCR purification kit, and DNA concentration was measured with Qubit 2.0 Fluorometer (Invitrogen). The PCR station and materials were cleaned with RNaseZap (Invitrogen) and 70% ethanol and UV-sterilized before use.

We used Next-Generation Sequencing (NGS, Illumina-MiSeq) and followed the Illumina Metagenomic Sequencing Library Preparation protocol (Illumina) for indexing, and other preparation. Sequencing was conducted on an Illumina platform (MiSeq, Pair-end sequencing with a length of 301 bp) at KAUST Biological Core Lab with the Illumina MiSeq Reagent kit v3 (600 cycles).

The Illumina MiSeq sequencing output was demultiplexed using the Illumina protocol and primers were trimmed using Cutadapt 1.17 with default settings (Martin, 2011).

Funding

Independent Research Fund Denmark, Award: 8021-00222 B