16S and 18S V4 rRNA metabarcoding sequencing of bacterial, archaeal, and eukaryote communities in benthic sediment sampled along a geographical gradient around a shrimp farm (Toahotu lagoon, French Polynesia) for the DEPOMOD-shrimp project, 2020-2024
To compare benthic bacterial, archaeal and metazoan communities (abundance, composition, etc.) around a shrimp farm (cages located in Toahotu lagoon, French Polynesia), sediment samples were collected around the farm and further south using a 0.1 m2 Van Veen grab. Twelve stations were located in the study area of the farm, from 0 to 200 m from the cages, and another station serving as a technical control was located several kilometers south of the farm (station X).  For archaeal and bacterial DNA extractions (16S-dataset), three replicates of sediment were collected in three (unsieved) grabs (Ntotal = 9) at each station in clean 15 mL Flacon tubes preserved on ice on the boat then frozen at -80C at the laboratory. After thawing on ice, ~ 3 g of wet sediment from each sample were transferred into PowerBead tubes, homogenised with 200 L TrisEDTA, and DNA was extracted following instructions from the DNeasy Power Soil kit (Qiagen, CA, USA). DNA concentrations were verified with NanoDrop ND2000 (Thermo-Fisher) and DNA solutions were dried in 96-well plates using a SpeedVac for 4 hours, and frozen at -20C. An amplicon library targeting the V4 region of the 16S rRNA gene for prokaryotes (~250300 base pairs) was built using the following primers: 515F: 5-GTG YCA GCM GCC GCG GTA A-3 (Parada et al. 2016) and 806R: 5-GGA CTA CNV GGG TWT CTA AT-3 (Apprill et al. 2015). For metazoan DNA extractions (18S-dataset), three replicates of the material retained on the 0.5 mm sieve (Section 2.5) were collected in three grabs (Ntotal = 9) at each station in clean 15 mL Flacon tubes preserved on ice on the boat then frozen at -80C at the laboratory. Using the sieved material allowed to focus on target benthic fauna (infauna, endofauna, meiofauna and macrofauna) to improve the DNA signal. After thawing on ice, ~3 g of wet sediment from each sample were transferred into 2 mL Eppendorf tubes and filled with distilled water, vortexed and centrifuged (5000 g, 24C, 2 min). The supernatant was removed and the same operations were performed one more time, which allowed proper desalination of the samples. The material was then inserted into PowerBead tubes, homogenised with 200 L TrisEDTA, and DNA was extracted following instructions from the DNeasy Power Soil kit (Qiagen, CA, USA). DNA preparation and sequencing followed the same procedure as described above, including extraction and PCR negative controls, after building a library targeting eukaryotes using the universal eukaryotic primers TAReuk454FWD: 5-CCA GCA SCY GCG GTA ATT CC-3 and TAReukREV3: 5-ACT TTC GTT CTT GAT YRA-3 (Hadziavdic et al. 2014) to amplify the V4 region (~380 base pairs) of the 18S rRNA gene. The libraries were then multiplexed (117 samples per run) and sequenced on an Illumina MiSeq platform using 250-bp paired-end reads at Genome Quebec (Montreal, Canada). Extraction and PCR negative controls were included throughout the workflow to assess potential contamination.
To compare benthic bacterial, archaeal and metazoan communities (abundance, composition, etc.) around a shrimp farm (cages located in Toahotu lagoon, French Polynesia), sediment samples were collected around the farm and further south using a 0.1 m2 Van Veen grab. Twelve stations were located in the study area of the farm, from 0 to 200 m from the cages, and another station serving as a technical control was located several kilometers south of the farm (station X).  For archaeal and bacterial DNA extractions (16S-dataset), three replicates of sediment were collected in three (unsieved) grabs (Ntotal = 9) at each station in clean 15 mL Flacon tubes preserved on ice on the boat then frozen at -80C at the laboratory. After thawing on ice, ~ 3 g of wet sediment from each sample were transferred into PowerBead tubes, homogenised with 200 L TrisEDTA, and DNA was extracted following instructions from the DNeasy Power Soil kit (Qiagen, CA, USA). DNA concentrations were verified with NanoDrop ND2000 (Thermo-Fisher) and DNA solutions were dried in 96-well plates using a SpeedVac for 4 hours, and frozen at -20C. An amplicon library targeting the V4 region of the 16S rRNA gene for prokaryotes (~250300 base pairs) was built using the following primers: 515F: 5-GTG YCA GCM GCC GCG GTA A-3 (Parada et al. 2016) and 806R: 5-GGA CTA CNV GGG TWT CTA AT-3 (Apprill et al. 2015). For metazoan DNA extractions (18S-dataset), three replicates of the material retained on the 0.5 mm sieve (Section 2.5) were collected in three grabs (Ntotal = 9) at each station in clean 15 mL Flacon tubes preserved on ice on the boat then frozen at -80C at the laboratory. Using the sieved material allowed to focus on target benthic fauna (infauna, endofauna, meiofauna and macrofauna) to improve the DNA signal. After thawing on ice, ~3 g of wet sediment from each sample were transferred into 2 mL Eppendorf tubes and filled with distilled water, vortexed and centrifuged (5000 g, 24C, 2 min). The supernatant was removed and the same operations were performed one more time, which allowed proper desalination of the samples. The material was then inserted into PowerBead tubes, homogenised with 200 L TrisEDTA, and DNA was extracted following instructions from the DNeasy Power Soil kit (Qiagen, CA, USA). DNA preparation and sequencing followed the same procedure as described above, including extraction and PCR negative controls, after building a library targeting eukaryotes using the universal eukaryotic primers TAReuk454FWD: 5-CCA GCA SCY GCG GTA ATT CC-3 and TAReukREV3: 5-ACT TTC GTT CTT GAT YRA-3 (Hadziavdic et al. 2014) to amplify the V4 region (~380 base pairs) of the 18S rRNA gene. The libraries were then multiplexed (117 samples per run) and sequenced on an Illumina MiSeq platform using 250-bp paired-end reads at Genome Quebec (Montreal, Canada). Extraction and PCR negative controls were included throughout the workflow to assess potential contamination.
The French Research Institute for Exploitation of the Sea