Introduction

Methods

Sampling

Sample collection was conducted under the standard operating procedures of the EMO BON Handbook (Santi et al. 2021). Water samples were collected and processed according to the "Water Column Standard Operating Procedures 1 ‒ WaSOP 1 (basic)": subsurface seawater was collected from the water column sampling site of each observatory, pre-filtered (> 200 μm) and concentrated by sequential filtration on polycarbonate (PC) membrane filters of 142 mm in diameter and pore sizes 3 µm and 0.2 µm. This resulted in two different plankton size fractions: 3-200 μm and 0.2-3 μm. After filtration, each filter membrane was cut into two pieces by a sterile scalpel and each filter piece was considered to represent one replicate. In total, four replicates were collected from each sampling, since two separate sequential filtrations were conducted at each sampling site. Subsequenctly, membranes (replicates 1 and 2) were placed in individual containers with the DNA/RNA shield preservative (Zymo Research), flash frozen in liquid nitrogen and stored at -80^oC until shipment to the sequencing facility. Filter membranes that were collected for biobanking (replicates 3 and 4) were preserved in cryotubes without the addition of DNA/RNA Shield and stored at -80^oC.

Sediment samples were collected and processed, based on all the three proposed protocols. Briefly, observatories NRMCB and RiaFormosa used the 'Soft Substrate Standard Operating Procedures 1 – SoSOP 1 (intertidal sediments)", while OOB and ROSKOGO used the "Soft substrate Standard Operating Procedures 2 – SoSOP 2 (coastal sediments by diving)" and BPNS used "Soft substrate Standard Operating Procedures 3 ‒ SoSOP 3 (coastal sediments by research vessel)". Regardless of the choice of protocol, the steps regarding collection of sediment for microbial community assessment include the use of sediment cores and the subsequent slicing of the top 5 cm layer. As for the water samples, four replicates are collected for the sediment sampling; DNA/RNA shield was added in two of the replicates, which were the ones shipped for sequencing.

Geographic range

The dataset's geographical range includes 14 locations (13 observatories) across eight ecoregions, based on the Marine Ecoregions of the World (MEOW), proposed by Spalding et al. (2007) (Table 1; Fig. 1); details are also provided regarding the locality of the observatories, from the broader (ocean/sea) to the regional and the local scale.

Table 1.

Download as

CSV

XLSX

Locality and coordinates of the sampling stations.

Observatory	Coordinates	Marine Ecoregion of the World (MEOW)	Ocean/Sea	Region	Location	Water sampling	Sediment sampling	Number of collected samples	Number of successfully sequenced samples
AAOT	45.31417 N; 12.508333 E	Adriatic Sea	Mediterranean Sea - Eastern Basin	Adriatic Sea	Gulf of Venice	Yes		8	7
BPNS	51.43333 N; 2.808331 E	North Sea	North Atlantic Ocean	North Sea	Belgian part of the North Sea	Yes	Yes	12	11
EMT21	42.20194 N; -8.798500 E	South European Atlantic Shelf	Atlantic Ocean	North Atlantic Ocean	Vigo Seamount	Yes		4	4
ESC68N	68.92589 N; 17.125619 E	Northern Norway and Finnmark	Arctic Ocean	Norwegian Sea	Norwegian part of the Norwegian Sea	Yes		4	4
HCMR-1	35.34662 N; 25.278761 E	Aegean Sea	Mediterranean Sea - Eastern Basin	Aegean Sea	Crete Sea	Yes		12	5
IUIEilat	29.50000 N; 34.916667 E	Northern and Central Red Sea	Indian Ocean	Gulf of Eilat	Gulf of Eilat	Yes		4	4
NRMCB	40.80014 N; 14.250000 E	Western Mediterranean	Mediterranean Sea - Western Basin	Tyrrhenian Sea	Naples Gulf	Yes	Yes	18	13
OOB	42.48417 N; 3.135278 E	Western Mediterranean	Mediterranean Sea - Western Basin	Gulf of Lion	Bay of Banyuls-sur-Mer		Yes	3	1
OSD74	41.14653 N; -8.666639 E	South European Atlantic Shelf	Atlantic Ocean	North Atlantic Ocean	Porto Valley	Yes		3	3
PiEGetxo	43.33858 N; -3.014639 E	South European Atlantic Shelf	North Atlantic Ocean	Bay of Biscay	Abra de Bilbao	Yes		6	6
RFormosa	37.00564 N; -7.969250 E	South European Atlantic Shelf	Atlantic Ocean	North Atlantic Ocean	Ria Formosa	Yes	Yes	6	6
ROSKOGO	48.70833 N; -3.866000 E	Celtic Seas	North Atlantic Ocean	English Channel	French part of the English Channel		Yes	2	2
ROSKOGO	48.77167 N; -3.968333 E	Celtic Seas	North Atlantic Ocean	English Channel	French part of the English Channel	Yes		8	6
VB	43.68300 N; 7.317000 E	Western Mediterranean	Mediterranean Sea - Western Basin	Villefranche Bay	Villefranche Bay - Point B	Yes		8	8

a

b

Figure 1.

Map of the observatories collecting:

a: sediment samples;  
b: water samples.  

DNA extraction, library preparation and sequencing

DNA extraction was performed at Genoscope, which was the chosen centralised facility to minimise biases and follow the same standardised procedures. For DNA extraction of the water column filter samples, the same protocol as described by Alberti et al. (2017) was used. The procedure consisted of a first step of cell disruption by cryogenic grinding of membrane filters followed by chemical lysis and then nucleic acid purification using NucleoSpin RNA kits, combined with the NucleoSpin RNA/DNA buffer set (Macherey-Nagel, Düren, Germany). For sediment samples, DNA extraction was performed using the commercially available DNeasy PowerSoil Pro Kit (Qiagen) with slight modifications.

Sequencing was also performed at Genoscope. Metagenome libraries were constructed according to the available DNA: 10 to 100 ng of genomic DNA were sonicated to obtain fragments of around 350 bp, using the Covaris E220 instrument (Covaris, Woburn, MA, USA). Fragments were repaired, 3'-adenylated and NEXTflex PCR freebarcodes adapters (Bioo Scientific, Austin, TX, USA) were added using the NEBNext® Ultra II DNALibrary prep kit for Illumina (New England Biolabs, Ipswich, MA, USA). Ligation products were purified by AMPure XP beads 0:8 volume (Beckmann Coulter, Brea, CA, USA). DNA fragments (> 200 bp) were amplified by PCR (2 PCR reactions, 14 cycles) using Illumina adapter-specific primers and NEBNext® Ultra II Q5 Master Mix (NEB). All libraries were subjected to size profile analysis conducted by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and to qPCR quantification using the KAPA Library Quantification Kit for Illumina Libraries (KapaBiosystems, Wilmington, MA, USA). All metagenomic libraries validated by the quality-control were sequenced using 151-bp pairwise read chemistry on an Illumina NovaSeq6000 sequencer, using S4 Flowcells (Illumina, San Diego, CA, USA). A minimum of 40,000 million useful paired-end reads were obtained per sample. Short Illumina reads were bioinformatically post-processed sensu Alberti et al. (2017) to filter out low-quality data. First, low-quality nucleotides (Q < 20) were discarded from both read ends. Then the remaining Illumina sequencing adapters and primer sequences were removed and only reads ≥ 30 nucleotides were retained. These filtering steps were done using in-house-designed software, based on the FastX package (Engelen and Aury 2016). Finally, read pairs mapping to the phage phiX genome were identifed and discarded using SOAP aligner (Li et al. (2008), default parameters) and the Enterobacteria phage PhiX174 reference sequence (GenBank: NC_001422.1).

Data Resources

Usage Rights

Acknowledgements

Conflicts of interest

References

Alberti A, Poulain J, Engelen S, Labadie K, Romac S, Ferrera I, Albini G, Aury J, Belser C, Bertrand A, Cruaud C, Da Silva C, Dossat C, Gavory F, Gas S, Guy J, Haquelle M, Jacoby E, Jaillon O, Lemainque A, Pelletier E, Samson G, Wessner M, Bazire P, Beluche O, Bertrand L, Besnard-Gonnet M, Bordelais I, Boutard M, Dubois M, Dumont C, Ettedgui E, Fernandez P, Garcia E, Aiach NG, Guerin T, Hamon C, Brun E, Lebled S, Lenoble P, Louesse C, Mahieu E, Mairey B, Martins N, Megret C, Milani C, Muanga J, Orvain C, Payen E, Perroud P, Petit E, Robert D, Ronsin M, Vacherie B, Acinas S, Royo-Llonch M, Cornejo-Castillo F, Logares R, Fernández-Gómez B, Bowler C, Cochrane G, Amid C, Hoopen PT, De Vargas C, Grimsley N, Desgranges E, Kandels-Lewis S, Ogata H, Poulton N, Sieracki M, Stepanauskas R, Sullivan M, Brum J, Duhaime M, Poulos B, Hurwitz B, Acinas S, Bork P, Boss E, Bowler C, De Vargas C, Follows M, Gorsky G, Grimsley N, Hingamp P, Iudicone D, Jaillon O, Kandels-Lewis S, Karp-Boss L, Karsenti E, Not F, Ogata H, Pesant S, Raes J, Sardet C, Sieracki M, Speich S, Stemmann L, Sullivan M, Sunagawa S, Wincker P, Pesant S, Karsenti E, Wincker P (2017)
Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition
.
Scientific Data
4
(
1
). https://doi.org/10.1038/sdata.2017.93
Courtot M, Gupta D, Liyanage I, Xu F, Burdett T (2021)
BioSamples database: FAIRer samples metadata to accelerate research data management
.
Nucleic Acids Research
50
https://doi.org/10.1093/nar/gkab1046
Engelen S, Aury J (2016)
fastxtend
. URL: https://www.genoscope.cns.fr/fastxtend/.
Li R, Li Y, Kristiansen K, Wang J (2008)
SOAP: short oligonucleotide alignment program
.
Bioinformatics
24
(
5
):
713
‑
714
. https://doi.org/10.1093/bioinformatics/btn025
Santi I, Casotti R, Comtet T, Cunliffe M, Koulouri P(, Macheriotou L, Not F, Obst M, Pavloudi C, Romac S, Thiebaut E, Vanaverbeke J, Pade N (2021)
European Marine Omics Biodiversity Observation Network (EMO BON) Handbook (Version 1.0).
EMBRC-ERIC
https://doi.org/10.25607/obp-1653
Santi I, Beluche O, Beraud M, Buttigieg PL, Casotti R, Cox C, Cunliffe M, Davies N, de Cerio OD, Exter K, Kervella AE, Kotoulas G, Lagaisse R, Laroquette A, Louro B, Not F, Obst M, Pavloudi C, Poulain J, Præbel K, Vanaverbeke J, Pade N (2023)
European marine omics biodiversity observation network: a strategic outline for the implementation of omics approaches in ocean observation
.
Frontiers in Marine Science
10
https://doi.org/10.3389/fmars.2023.1118120
Spalding M, Fox H, Allen G, Davidson N, Ferdaña Z, Finlayson M, Halpern B, Jorge M, Lombana A, Lourie S, Martin K, McManus E, Molnar J, Recchia C, Robertson J (2007)
Marine ecoregions of the world: A bioregionalization of coastal and shelf ereas
.
BioScience
57
(
7
):
573
‑
583
. https://doi.org/10.1641/b570707
Yuan D, Ahamed A, Burgin J, Cummins C, Devraj R, Gueye K, Gupta D, Gupta V, Haseeb M, Ihsan M, Ivanov E, Jayathilaka S, Kadhirvelu VB, Kumar M, Lathi A, Leinonen R, McKinnon J, Meszaros L, O'Cathail C, Ouma D, Paupério J, Pesant S, Rahman N, Rinck G, Selvakumar S, Suman S, Sunthornyotin Y, Ventouratou M, Vijayaraja S, Waheed Z, Woollard P, Zyoud A, Burdett T, Cochrane G (2024)
The European Nucleotide Archive in 2023.
Nucleic acids research
52
(
D1
):
D92-D97
. https://doi.org/10.1093/nar/gkad1067

Supplementary materials