Résumé
This dataset presents the diversity of the marine microbiome based on samples of water collected during the ABRACOS 1 expedition in the Tropical Western Atlantic Ocean (2015-09-29 to 2015-10-21). The sampling was conducted along the northeastern margin of Brazil (approximately 3°S to 9°S and 35°W to 31°W), encompassing the continental shelf, the Fernando de Noronha archipelago, and Rocas Atoll.
Sample Collection :
Seawater samples were collected at different depths according to the fluorescence maximum using a 10L sterile Niskin bottle mounted on a CTD-Rosette. Planktonic prokaryotes (at least 500 mL of 20 µm pre-filtered) were collected on 0.22 μm Sterivex-GP (Millipore®), and the pressure filter units membranes were then placed at -80 °C until further analysis.
DNA extraction and PCR amplification :
Total genomic DNA GTTP filters was extracted using the MagAttract PowerSoil® DNA kit according to the manufacturer’s instructions (Qiagen, Courtaboeuf, France) with automated processing and the liquid handling system KingFisher FlexTM (ThermoScientific®, Waltam, MA, USA). Nucleic acids were eluted in molecular water (Merck MilliporeTM, Burlington, MA, USA) and quantified on a NanoDrop 8000 TM spectrophotometer (ThermoScientific®, Wilmington, MA, USA). The DNA extracts were stored at -20 °C until further analyses and PCR amplification. The V4-V5 region of the 16S rDNA gene was targeted with the universal primers 515F–Y (5′-GTGYCAGCMGCCGCGGTAA-3′) and 926R (5′-CCGYCAATTYMTTTRAGTTT-3′) (Parada et al., 2016) coupled with platform-specific Illumina adaptor sequences on the 5′ ends. Each 50 μL PCR reaction was prepared with 25 μL Taq Polymerase Phusion® High- Fidelity PCR Master Mix with GC Buffer (New England Biolabs®, Inc., Ipswich, MA, USA), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 2 μL template DNA, 1.5 μL DMSO, and 19.5 μL molecular water. PCR amplifications involved the following protocol: an initial 98 °C denaturing step for 30 s followed by 35 cycles of amplification (10 s denaturation at 98 °C; 1 min at 60 °C annealing; 1.5 min extension at 72 °C), and a final extension of 10 min at 72 °C. Amplification and primer specificity were verified by electrophoresis on a 2.0 % agarose gel for confirmation of ~450 bp amplicon size. Extraction of blank samples used as DNA extraction controls and standard mock communities (ZymoBIOMICS Microbial Community DNA Standards II, Zymo Research) was performed to evaluate the quality of our sample-processing pipeline. Sequencing was performed on Illumina Miseq by GeT-Biopuces (INSA, Toulouse, France).
Sequence processing :
Raw sequencing data was analyzed in R (version 4.1.1), using the dada2 pipeline (Callahan et al., 2016). Briefly, sequences were first trimmed and filtered based on read-quality profiles (maxN = 0; maxEE = [2,2]; truncQ = 2; and truncLen = [250,250]). Amplicon sequence variants (ASVs) were inferred using the dada2 algorithm (Divisive Amplicon Denoising Algorithm) after pooling dereplicated reads from all samples. Then forward and reverse reads were merged and chimeric sequences were removed. The taxonomic classification of ASVs was performed with the naive Bayesian RDP classifier implemented in dada2 and using the SILVA reference database nr_V132. The ASV count table, taxonomy and sequences were organized in a phyloseq object using the phyloseq package (v.1.28.0, McMurdie and Holmes, 2013) in R. We used a combination of two methods to remove contaminants from our dataset. First, the R package decontam (Davis et al., 2018) was used to identify ASV contaminants from the dataset based on the “prevalence method” of the package. However, some known extraction kit contaminants listed by Salter et al. (2014), such as ASVs from the genera Bradyrhizobium and Cupriavidus, remained in our dataset. They were manually removed from the final dataset. Taxonomic diversity of each microbial community was measured using richness (number of ASVs).
Dataset organization :
(Both files are in csv format, with decimal point and semicolon separator. Their structure is detailed in a ReadMe file)
- Microbiome_ASV_Reference_List_Abracos1.csv : the 4434 different ASVs (Amplicon Sequence Variants) with their taxonomy (Kingdom, Phylum, Class, Order, Family, Genus)
- Microbiome_ASV_Occurrence_Abracos1.csv : a table in long format giving the occurrence of the 4434 ASVs in the 46 samples, with information on the samples. (203964 rows)
- Readme_Microbiome_Abracos1.txt : ReadMe file describing the structure of the 2 data files