|Created||August 16, 2016|
|Modified||August 22, 2016|
|State||Preliminary and in progress|
This is the manually curated genome of the SAG SCGC AD-561-N23
Samples for molecular biological analysis were collected shipboard from 10 cm sections of the intact core using sterile cut-end 5 mL syringes. Samples were immediately frozen at –80 degrees C and maintained at this temperature during transport and storage to the home laboratory. Sorting of individual cells from unfixed frozen sediment from 97.41 mbsf was attempted for single cell genomics using the Single Cell Genomics Center (SCGC) at the Bigelow Laboratory for Ocean Sciences. While the sediment was not preserved with recommended fixatives to prevent cell lysis during storage, and although the samples had gone through at least one round of freeze-thaw for bulk DNA extraction which may have introduced cell lysis, the high relative abundance of target microorganisms in the sample suggested this approach might still be successful in recovering intact cells. Approximately 0.5 g of frozen sediment was diluted in 1 mL of filter-sterilized seawater and vortexed for 30 s to liberate cells from the sediment matrix, modifying methods developed previously. Sediment was then separated from cells by gentle centrifugation at 2000 rpm for 30 s The cell suspension was treated with SYTO-9 DNA stain and sorted into two 384-well plates using SCGC’s s tandard pipeline. Both sorted plates were subjected to physical lysis treatments (five freeze- thaw cycles), and the second plate also experienced an alkaline lysis treatment. DNA amplification by multiple displacement amplification.
The initial assembly of Atribacteria bacterium SCGC AD- 561-N23 is publically available within the IMG system (taxon ID 2588254308) and the sequence for the 16S rRNA gene is available within the IMG system and Supplementary Materials. A detailed assembly procedure (QC.finalReport.pdf ) can be downloaded from: http://genome.jgi.doe.gov/CandivSCAD561N23/CandivSCAD561N23.download.html.
Briefly, single-cell amplified genomic (SAG) DNA was sequenced, assembled and annotated at the United States Department of Energy’s Joint Genome Institute (JGI) following their standard pipeline for Illumina HiSeq 2000 platform sequencing. Illumina reads were screened using JGI’s in-house DUK filtering program (Mingkun et al., unpublished). Trimmed reads were assembled using SPAdes (version 3.0.0) with the following parameters (–t 8 –m 40 – –sc – –careful – –12; Bankevich et al., 2012). Once released to Integrated Microbial Genomes (IMG) system, manual screening and removal of potential contaminate sequences according to JGI’s single cell data decontamination protocol (Clingenpeel, 2015). Scaffolds with GC contents that varied from the genome average more than 10% and clustered as a distinct group according to a kmer analysis (IMG, fragment window 5000 bp, fragment step 500 bp, oligomer size 5, minimum variation 10) were identified as potential contaminates and were removed from the de novo assembly (with the exception of scaffolds that contained ribosomal DNA). This screened genome was submitted to the IMG database as GOLD project Gp0087948, titled “Candidate division JS 1 bacterium SCGC AD-560-N23 (manually screened)”. Gene annotations were performed using both IMG and the Rapid Annotation using Subsystem Technology (RAST) platforms (Aziz et al., 2008; Overbeek et al., 2013 ;Markowitz et al., 2014). Discrepancies between annotations were investigated by comparing coding sequences of genes against GenBank non-redundant protein sequence and Swiss- Prot Databases by BLASTP (Altschul et al., 1990). Genome completeness was estimated by comparing the annotated genome sequence against a list of conserved single copy bacterial genes.
This screened genome was submitted to the IMG database as GOLD project Gp0087948 with an IMG taxon ID of 2626541500, and to MG-RAST under accession numbers 4624791.3–4634830.3.
|Stephanie A. Carr||Colorado School of Mines (CSM)||✓|
|Beth N. Orcutt||Colorado School of Mines (CSM)||✓|
|John R. Spear||Bigelow Laboratory for Ocean Sciences|
|Shannon Rauch||Colorado School of Mines (CSM)|
|Shannon Rauch||Colorado School of Mines (CSM)|
|Shannon Rauch||Woods Hole Oceanographic Institution (WHOI BCO-DMO)|
BCO-DMO Project Info
|Project Title||Functional potential of the uncultivated Candidate Phylum OP9 from deep Antarctic marine sediments using single cell genome techniques|
|Acronym||Adelie Basin Atribacteria|
|Created||November 20, 2015|
|Modified||November 20, 2015|
Bacteria belonging to the newly classified candidate phylum “Atribacteria” (formerly referred to as “OP9” and “JS1”) are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica’s Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.
This project was funded by a C-DEBI Postdoctoral Fellowship