DOI: 10.5061/dryad.1jwstqjw9
title: Raw cutadapt miseq output - minibarcode 18S-V7 of macroalgae

description: 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.
description: 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).