Dataset: DHAB halocline sediment pyrotags - prokaryotes
Deployment: AT18-14

RNA was purified using the MEGAclear kit (Ambion, USA). Reverse transcription of the purified RNA samples was performed using the QuantiTect kit (Qiagen, USA). Tag-pyrosequencing of the 16S rRNA gene was performed using PCR amplification of the V4-V6 region of the 16S rRNA gene and the primer pair S-DBact-0341-b-S-17 (5’-CCTACGGGNGGCWGCAG-3’) and S-D-Bact-0785-a-A-21 (5’-GACTACHVGGGTATCTAATCC-3’) for Bacteria (Klindworth et al. 2012), and archaea349F (5’-GYGCASCAGKCGMGAAW-3’) and archaea806R (5’-GGACTACVSGGGTATCTAAT-3’) for Archaea (Takai & Horikoshi 2000), as described in Dowd et al., (2008). A one-step, 30-cycle PCR reaction was performed using HotStarTaq Plus Master Mix Kit (Qiagen, USA).  Following PCR, all amplicon products (ca. 450 bp) from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, USA). Samples were sequenced utilizing the Roche 454 FLX Titanium platform and reagents according to the guidelines at the MRDNA Ltd. (Shallowater, TX, USA) sequencing facility. Processing of the resulting sequences, i.e. trimming and quality control, was performed with the MOTHUR software (v 1.30)  including denoising of the flowgrams using PyroNoise and data normalization to the smallest number of sequences in the resulting libraries. Sequences ≥250 bp with no ambiguous base assignments and no homopolymers ≥8 bp were included in downstream analyses. Single singletons, i.e. sequences that appeared only with once in the whole dataset, were excluded from further analysis. These sequences were aligned using the SILVA SSU database (release 108). All sequences were binned into Operational Taxonomic Units (OTUs) and were clustered (average neighbor algorithm) at 97% sequence similarity. Coverage values were calculated with MOTHUR (v 1.30).

BCO-DMO Processing:
- original file: EdgcombDHABprokaryoteTags.xlsx
- replaced positions for Discovery and Urania with those in event log for pushcores
- added html links to GenBank BioProject
- added cruise_id, lat, lon