COI Metabarcoding Genetic Observations of Marine Species in the Port of Wallhamn, Sweden (2022)

Study Extent

The different sampling methods had different timings over the summer 2022: The eRAS panels were put out on the 23rd of June and taken back on the 9th of September, one was in the industrial port, three were in the marina. The artificial habitats were put out on the 23rd of June and taken back on the 4th of August, one in each area. The ARMS were put out on the 23rd of June and taken back on the 9th of September, one in the industrial port and two in the marina. In the marina, three plankton samplings occurred on the 23rd of June, the 4th of August and the 9th of September (9 plankton samples in total). Similarly, in the marina, three eDNA samplings occurred on the 23rd of June, the 4th of August and the 9th of September (9 eDNA samples in total). Three scraping/RAS samplings occured on the 9th of September in the marina.

Sampling

Artificial habitats are plastic trays filled with pottery shards, a weight at the bottom to hold the trays down and a net above to keep the shards in place. On two sides, larger holes were cut to allow fish to move freely in and out of the habitat Plankton samples were taken as vertical hauls from the bottom (5-10 m) up to the surface with a 90 µm plankton bucket. The samples were fixed in 95% ethanol directly in place with an amount of alcohol that was judged to result in a sample with at least 70% ethanol. Upon return to the lab, the plankton samples were decanted and new alcohol was added to ensure that the ethanol content was sufficiently high. The samples were then stored in a freezer (-20°C) until extraction. Water samples for eDNA were taken with Ruttner retrievers at slightly different depths on the premises and pooled in a 1 litre vessel. Water was filtered on site and fixed with 95% ethanol.The filters were stored in a freezer (-20°C) until extraction.

Quality Control

Negative controls (blank samples) were created by the same extraction procedure but without the sampled material. No DNA could be detected in these samples, with neither Nanodrop nor Qubit. The blank samples were included in the preparation of libraries (below) as a further control for contamination during the extraction of DNA. Another negative control was performed during PCR.

Method steps

1. DNA was extracted from the filters with the Nucleospin eDNA water kit (Macherey-Nagel) using the Nagel) using the technique and standard developed in the laboratory. Concentration of DNA in the extracts was measured with the Qubit® fluorometer. Note that this measures concentration of all DNA in the sample and not specific DNA from the target species. This is done to ensure that the extractions have worked.
2. The genetic analysis and comparison was based on two molecular markers (COI and 18S). These markers were amplified for each sample. Same PCR protocols were used for metabarcoding of the ARMS, eDNA and plankton samples The DNA libraries were sequenced with Illumina MiSeq, 2 x 300 bp.
3. The analysis was performed in the R-environment with a DADA2 package. After initial quality control, the sequences were filtered for low quality, as well as adapters and primers were removed. Sequencing errors were corrected by calculating an error model; and after that singletons and chimeras were removed using the “pseudo-pooling” function in DADA2.
4. The filtered and quality-controlled COI sequences were matched against the BOLD database with the python package BOLDigger. The 18S sequences were matched against the PR2 database. All subsequent analysis was performed with customized R scripts where identified species were matched against different NIS and IAS species reference lists.

N/A

1. Mollusca
   rank: phylum
2. Hydrozoa
   rank: class
3. Arthropoda
   rank: phylum
4. Diptera
   rank: order
5. Dinophyceae
   rank: class
6. Calanoida
   rank: order
7. Pectinariidae
   rank: family
8. Campanulariidae
   rank: family
9. Chromista
   rank: kingdom
10. Ostreida
    rank: order
11. Gonyaulacales
    rank: order
12. Polychaeta_X
    rank: order
13. Cephalaspidea
    rank: order
14. Animalia
    rank: kingdom
15. Canidae
    rank: family
16. Phyllodocida
    rank: order
17. Gastropoda_X
    rank: order
18. Venerida
    rank: order
19. Annelida
    rank: phylum
20. Polychaeta
    rank: class
21. Littorinimorpha
    rank: order
22. Unassigned
    rank: kingdom
23. Chordata
    rank: phylum
24. Gonyaulacales_X
    rank: family
25. Phoronida_X
    rank: class
26. Insecta
    rank: class
27. Veneridae
    rank: family
28. Cerithiidae
    rank: family
29. Phoronidae
    rank: family
30. Bivalvia
    rank: class
31. Carnivora
    rank: order
32. Copepoda
    rank: class
33. Oithonidae
    rank: family
34. Chironomidae
    rank: family
35. Philinidae
    rank: family
36. Phoronida_XX
    rank: order
37. Myzozoa
    rank: phylum
38. Mammalia
    rank: class
39. Spionidae
    rank: family
40. Ostreidae
    rank: family
41. Phoronida
    rank: phylum
42. Hydrobiidae
    rank: family
43. Cyclopoida
    rank: order
44. Leptothecata
    rank: order
45. Nereididae
    rank: family
46. Centropagidae
    rank: family
47. Gastropoda
    rank: class
48. Cnidaria
    rank: phylum
49. Acartiidae
    rank: family

This dataset covers the port of Wallhamn (Sweden) which has both a commercial/industrial part in the North and a marina in the South.