|Created||January 18, 2016|
|Modified||August 19, 2016|
|State||Final no updates expected|
Whole genome sequence data from bacterial isolates from venting fluids at NW Rota Seamount, collected in 2009 and 2010.
Diffuse hydrothermal vent fluids were collected at several vent sites on NW Rota-1 seamount in 2009 and 2010 using the ROV Jason 2 and the hydrothermal fluid and particle sampler. Anaerobic enrichment media previously used for the isolation of Caminibacter profundus was inoculated with 1 ml of unfiltered diffuse flow fluids and incubated at 55 degrees C. Enrichments with positive microbial growth were isolated by three sets of dilution-to-extinction. The growth of Lebetimonas under varying conditions including alternative electron donor/acceptor pairs and with N2 gas as the sole nitrogen source was evaluated as described in the Supplementary Material of Meyer & Huber (2014). Growth of Lebetimonas strain JH369 with N2 gas as the sole nitrogen source was evaluated using anaerobic seawater media without yeast extract or ammonia and containing formate and elemental sulfur with an 80% N2 and 20% CO2 headspace.
Genomic DNA was extracted from pure cultures at log phase using a CTAB extraction. Libraries were prepared using Nextera DNA sample prep kits (Illumina, San Diego, CA, USA) and sequenced by Roche 454 GS FLX Titanium (454 Life Sciences, Branford, CT, USA) and/or using Illumina HiSeq 2000 paired reads (Illumina). In the case of strains sequenced with multiple platforms, the same genomic DNA extraction was used for all library preparations, with the exception of strain JS085. Genomes were assembled using several tools as described in the Supplementary material of Meyer and Huber 2014.
Meyer, J.L. and J.A. Huber. 2014. Strain-level genomic variation in natural populations of Lebetimonas from an erupting deep-sea volcano. ISME Journal. 8:867–880. doi:10.1038/ismej.2013.206
Prior to assembly, Illumina sequences were quality filtered using adaptive window trimming and a quality threshold of 30 using the script Trim.pl (http://wiki.bioinformatics.ucdavis.edu/index.php/Trim.pl). All reads were screened for adaptor, barcorde, primer, and transposan sequences and trimmed as needed using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). De novo genome assembly was performed with several assembly programs. Sequences generated through the 454 platform were first assembled with Roche’s GS De Novo Assembler v 2.6 (“Newbler”) 2 using default parameters. De novo assemblies of 454 reads were also performed using mira 3 with the default settings for normal quality de novo genome assembly. De novo assembly of subsets of Illumina reads was performed with velvet 4, using an estimated coverage of 1000x, kmer size of 21, and a coverage cutoff of 5). Large contigs from Newbler , mira, and velvet were consolidated using Geneious Pro v 5.6.6 (Biomatters, Ltd, http://www.geneious.com) and aligned with progressiveMauve 5 to visualize the relationship of large contigs from different assemblies and to identify gaps to close. Primers were designed at the ends of contigs using either Geneious Pro or CLC Genomics Workbench v 5.1 (CLCbio, http://www.clcbio.com) to amplify gaps between contigs. Positive PCR amplification products linking contigs were cleaned using a Min-Elute PCR Purification kit (Qiagen) and Sanger sequenced. A nearly complete draft genome from strain JS085 served as a reference genome for the remaining five strains. Both Illumina and 454 reads were mapped to the reference genome with CLC Genomics Workbench. Unmapped reads were then assembled de novo to ensure that novel genomic content in the mapped strains was not overlooked. De novo assembly of 454 and/or Illumina reads for each strain was also performed in CLC Genomics Workbench and compared to the mapped assemblies using progressiveMauve.
Four of the strains were sequenced using both 454 and Illumina and two strains were sequenced only with Illumina. The sequencing coverage depth of quality-filtered reads ranged from 22X to 50X for 454 and up to 3618X for Illumina. Lebetimonas strain JS085 had the highest coverage of 454 reads and was assembled into 33 large contigs with Newbler and 1747 contigs with mira. The 20 largest contigs from each of these assemblies were consolidated using de novo assembly in Geneious to 10 contigs. An additional round of assembly in Geneious with the 10 consolidated contigs and velvet contigs greater than 10 Kbp further consolidated the draft genome to 6 contigs. Primers were designed for all possible combinations between the 6 contigs. One gap was closed using Sanger-sequenced positive pcr products. Finally, all 454 and Illumina reads for strain JS085 were mapped to the draft genome consisting of 5 contigs and the resulting consensus was used as the final draft genome. The five remaining genomes were assembled by mapping 454 and Illumina reads to the JS085 reference genome in CLC Genomics Workbench. Hybrid de novo assemblies in CLC Genomics Workbench of each strain did not extend contigs or close gaps between the 5 contigs of the draft genomes. Assemblies of unmapped reads produced only short contigs with no significant similarities using nucleotide BLAST 6.
– modified parameter names to conform with BCO-DMO naming conventions;
– added hyperlinks;
– removed “m” (meters) in depth column.
Libraries were prepared using Nextera DNA sample prep kits (Illumina, San Diego, CA, USA) and sequenced by Roche 454 GS FLX Titanium (454 Life Sciences, Branford, CT, USA) and/or using Illumina HiSeq 2000 paired reads (Illumina).
General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.
phylum, organism taxonomic level. May include super-phylum and sub-phylum
order, organism taxonomic level; may include super-order and sub-order
Family. One of the levels in the taxonomic system of classification; typically ends in 'ae'. . May include super-family and sub-family.
a taxonomic binomial that consists of a genus name followed by the species name of an organism
Observation/sample depth below the sea surface. Units often reported as: meters, feet.
When used in a JGOFS/GLOBEC dataset the depth is a best estimate; usually but not always calculated from pressure; calculated either from CTD pressure using Fofonoff and Millard (1982; UNESCO Tech Paper #44) algorithm adjusted for 1980 equation of state for seawater (EOS80) or simply equivalent to nominal depth as recorded during sampling if CTD pressure was unavailable.
latitude, in decimal degrees, North is positive, negative denotes South; Reported in some datasets as degrees, minutes
longitude, in decimal degrees, East is positive, negative denotes West; Reported in some datsets as degrees, minutes
|Julie A. Huber||Marine Biological Laboratory (MBL)||✓|
|Shannon Rauch||Marine Biological Laboratory (MBL)||✓|
BCO-DMO Project Info
|Project Title||Functional gene diversity and expression in ocean crust microbial communities|
|Acronym||NP Functional Gene Div|
|Created||February 1, 2016|
|Modified||February 1, 2016|
Project description from C-DEBI:
The objective of this project is to determine the diversity, phylogeny, and expression of functional genes involved in carbon, hydrogen, and sulfur cycling in North Pond crustal fluids. These formation fluids are expected to be representative of the ubiquitous cold ocean crust habitat, where reactions between the water and mineral rock surfaces create substrates suitable for sustaining a potentially large reservoir of microbial life. Information regarding crustal microbial communities and the energy sources available for microbial metabolism has been limited by the inaccessibility of samples. IODP Expedition 336 will provide a unique opportunity to access deep subsurface formation fluids from North Pond, including sampling from multiple depth horizons within oceanic crust. My goal is to develop quantitative polymerase chain reaction assays to determine the expression of functional genes in order to increase our understanding of microbial metabolisms in deep subsurface environments.
This project was funded by a C-DEBI Postdoctoral Fellowship to Julie Meyer (formerly at the Marine Biological Laboratory).