Dataset: Sediment Bacteria Accession Numbers
Deployment: NH1212

Submarine Canyon Sediment Bacteria:

A ~35cm multicore was collected from La Jolla submarine canyon and sectioned using pre-sterilized tools shipboard at 1cm intervals for preservation at -80 deg C. DNA was extracted from ~2g subsamples using the MoBio PowerSoil DNA kit with an additional heating step applied during lysis. Sequence libraries were assembled for select bulk sediment samples for the v1–v3 region of the 16S rRNA gene using Eubacteria-specific primers (Muyzer et al. 1993) on the Illumina MiSeq platform (Bartram et al. 2011) at the University of Minnesota Genomics Center (UMGC).

Lake Surface Sediment Bacteria (excerpt from Harrison et al., 2016):

A 1.5 m hollow steel wedge was filled with a mixture of dry ice pellets and isopropyl alcohol and then lowered into the sediment. The coring device was held in place for 15 min and then raised back to the surface, where the dry ice slurry was poured off and unfrozen mud scraped off the sides. The device was then filled with lake surface water, and the frozen core slabs levered off the sides. The recovered frozen cores were wrapped in aluminum foil and placed on dry ice for transport. In the laboratory, the cores were placed on cardboard and kept frozen by regular contact with dry ice pellets. The sampling plane (facing away from the coring device) was smoothed down using a hand-held electric wood planer and utility knives until undisturbed laminae were clearly resolved. Core sections were selected by visual inspection on the basis of lamina thickness and continuity across the sampling plane.

Laminae were excised with pre-sterilized utility knife blades from the top 9 cm of the Twin Lake freeze core: five corresponding to white layers deposited during spring/summer blooms and four corresponding to winter deposition of organic matter and terrigenous particles. 0.2 g of material was collected from the sampling plane as described above, beginning at the lamina base and removing material no greater than 1 mm above that level.

From the Lake McCarrons freeze core, five spring/summer laminae were collected from sub-sampled portions of the 16–24 cm depth range by sequentially exposing the z-plane surface of the horizon with sterilized utility knife blades.”

Additional 5 mm frozen sediment wedges cross-cutting sediment laminae were taken at 2 cm intervals between 0 cm and 30 cm depth and 5 cm intervals from 30 cm to 65 cm, using pre-sterilized instruments. Subsamples were stored at -80 deg C for later extraction. An additional ambient-temperature piston core was collected from Lake McCarrons sediment at a position roughly adjacent (within 5 m) to the freeze core site and subsampled on shore within 2 h after removal. The presence and vertical migration of gas bubbles was observed in core sediment. 2 cc sediment aliquots were collected at 10 cm depth intervals starting at 2 cm beneath the sediment-water interface through predrilled windows, excluding the exterior 1 cm of material in contact with the coring tube. Subsamples were stored at -80 deg C.

DNA extraction, amplification and sequencing

Depth intervals corresponding to ambient core subsamples and excised laminae were used to select among the high-frequency bulk sediment samples of the McCarrons core. DNA was extracted from all sediment subsamples with a MoBio Soil DNA kit with an additional heating step added to the lysing step of manufacturer protocols (Harrison and Orphan 2012).

Sequence libraries were assembled for select bulk sediment samples for the v1–v3 region of the 16S rRNA gene using Eubacteria-specific primers (Muyzer et al. 1993) on the Illumina MiSeq platform (Bartram et al. 2011).