Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Metatranscriptome of whole microbial community from upper and lower halocline water column of Thetis DHAB

Related Reference:

Pachiadaki, M.G., Yakimov, M.M., Leadbetter, E., Edgcomb, V.P. 2014. Unveiling microbial activities along the halocline of Thetis, a deep hypersaline anoxic basin in the Eastern Mediterranean Sea. The ISME J. doi:10.1038/ismej.2014.100.

Water column samples were collected with a 12 L Niskin bottles housed on a rosette (General Oceanics) equipped with a SBE- 911plus conductivity-temperature-depth (CTD) sensor (Sea- Bird Electronics). Determination of oxygen concentration at selected depths was carried out using the Winkler method with an automatic endpoint detection burette (716 DNS Titrino, Metrohm AG). Water from two distinct horizons (each representing a distinct range of salinity and oxygen between the top and bottom of each Niskin bottle) was collected from the Thetis interface; the upper interface (UI) layer corresponding to 7.0 – 16.3% salinity, and the lower (LI) layer with 21.4 – 27.6% salinity. Oxygen in the UI sample ranged from 9.5 μmol L-1 to undetectable, and remained undetectable in the lower sample. From each horizon, ca. 12 L of water were collected on Durapore membranes (47 mm, 0.65 μm, Millipore) under gentle pressure (~100 ml min−1), using a peristaltic pump (Ecoline ISM 1079). The filters were stored in RNA Shield at -80oC until analysis. The filters were transferred into Lysing Matrix E tubes (MP Biomedicals). Four ml of RNeasy Midi Kit Buffer RTL (Qiagen) were added, homogenized for 60 seconds at 4.0 m/s using a FastPrep®-24 (MP Biomedicals), and centrifuged for 10 minutes at 4,000 x g. Liquids were transferred to clean tubes, 1 volume of 70% ethanol was added to each tube, and extracts were processed following the RNeasy kit instructions. Extractions were treated with DNase and purified with the MEGA Clear kit as described above. Fluid and filter extracts were combined for each sample and concentrated by ethanol precipitation. Absence of DNA was confirmed by 40 cycles of PCR using the general bacterial SSU rRNA gene primer 8F and the universal primer 1492R. For each extraction of total RNA (two replicates per depth horizon), cDNA was synthesized using the Ovation RNA-Seq System V2 Kit (NuGEN) following the manufacturer's instructions. Finally, cDNAs were purified with the MinElute Reaction Cleanup Kit (Qiagen) and sent for paired-end sequencing. One lane of Illumina HiSeq 2x100bp was requested for each sample (two replicates per lane). 

Forward and reverse reads were paired and filtered for quality control using CLC Genomics Workbench 5.0 (CLCBio) and a minimum quality score of 28, a minimum read length of 94 bp, allowing no sequences with ambigious nucleotides. The same platform was used to perform assembly of contigs and mapping of reads to contigs. The Rapid Analysis of Multiple Metagenomes with a Clustering and Annotation Pipeline was used through the CAMERA platform  to assign contigs to clusters of orthologous gene (COG) families, gene ontologies (GO), and protein families (Pfam) using four translation tables.

BCO-DMO Processing:
- original file: EdgcombThetisMetatranscriptomeData_final.xlsx
- added conventional header with dataset name, PI name, version date
- renamed parameters to BCO-DMO standard
- added cruise id, lat, lon
- added html links to GenBank BioProject

Protists are an essential component of microbial food webs and play a central role in global biogeochemical cycles, and thus are key players in sustaining the healthy functioning of any ecosystem. Over the past few years a rich diversity of protists has been revealed in a range of extreme environments, indicating that the frontiers of eukaryotic life are still being explored. Only recently, one of the most extreme marine environments known to science was discovered in the eastern Mediterranean Sea at a depth of ~3500m, namely deep hypersaline anoxic basins (DHABs). These basins are characterized by extremely high salt concentrations (up to saturation) that have been considered anathema to life. Instead, highly diverse communities of bacteria exist in the waters of these basins. With the exception of a preliminary study to this proposal that indicated a diverse and active assemblage of protists in the water column along the halocline and below the halocline, these DHABs remain largely unexplored regarding eukaryotic life forms. The sediments of the DHABs have not been explored for protists at all.

The investigators will collect water column and sediment samples on a short cruise to two basins with different brine chemistries. An exciting combination of molecular, cultivation-independent and culture-based approaches will be used to study the microbial communities of two basins. Investigators will use those approaches to determine adaptive strategies of marine protist communities to hypersaline, anoxic environments and the degree of their potential impact on biogeochemical cycling as a result of their predation activities, the degree to which the dominant protists maintain bacterial or archaeal symbionts, and the identity of those symbionts. The original research proposal identified Bannock and Discovery Basins as the field study areas, however the 2009 cruise collected samples at Discovery and Urania Basin. Methods to be employed include RNA-based sequence analysis of diversity based on 18S rDNA genes, statistical analyses of community composition and phylotype richness, geochemical documentation of the water column and sediments using classical and microelectrode approaches, expression profiling using 3'-UTR fragments of mRNAs, sequencing of complete gene transcripts for proteins appearing to confer adaptation to hypersalinity, analysis of the proteome signatures, FISH-SEM to characterize novel extremophiles, CARD-FISH to identify eukaryote prey and putative symbionts, and TEM to assess morphology and endobiont presence in common benthic morphotypes.

Hypersaline environments rank highly in the list of extreme systems that have attracted increasing notice in science as well as by the lay public. For example, considering predictions of increasing temperatures and drought in certain regions of our planet, the number of hypersaline habitats may increase dramatically causing this ecosystem to gain importance on a global scale. Thus, an understanding of the ecosystem in these habitats will help predict future ecosystem functioning due to global change. From a different perspective, revealing the mechanisms of adaptation to high salinity has become a major objective, both for biological science and for potential commercial exploitation of natural products associated with those adaptations.