Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Supplemental Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

These data were published in Hu et al., 2016.
Sequence data can be found in the NCBI SRA database under accession number SRP070577 with the associated BioProject PRJNA311248.

Samples were collected seasonally at the San Pedro Ocean Time-series (SPOT) station at four depths (surface, subsurface chlorophyll maximum, 150 and 890 m). The SPOT station was sampled from 5 m, the subsurface chlorophyll maximum (SCM), 150 and 890 m using 10 L Niskin bottles mounted on a CTD rosette, during regularly scheduled cruises (https://dornsife.usc.edu/spot/).

Seawater from all samples was sequentially pre-filtered through 200 μm and 80 μm Nitex mesh to reduce abundances of multicellular eukaryotes (metazoa). Near-surface and SCM seawater (2 L) and 150 and 890 m seawater (4 L) was filtered onto GF/F filters (nominal pore size 0.7 μm; Whatman, International Ltd, Florham Park, NJ, USA) and immediately flash frozen in liquid nitrogen for later DNA and RNA extraction.

Total DNA and RNA were extracted simultaneously from each sample using the All Prep DNA/RNA Mini kit (Qiagen, Valencia, CA, USA, #80204). Genomic DNA was removed during the RNA extraction with RNase-Free DNase reagents (Qiagen, #79254). Total extracted RNA was checked for residual genomic DNA by performing a polymerase chain reaction (PCR) using DNA specific primers to ensure that no amplified products appeared when run on an agarose gel. RNA was reverse transcribed into cDNA using iScript Reverse Transcription Supermix with random hexamers (Bio-Rad Laboratories, Hercules, CA, USA, #170-8840).

The resulting cDNA and DNA from each sample were PCR amplified using V4 forward (5′ -CCAGCA[GC]C[CT]GCGGTA ATTCC-3′ ) and reverse (5′ -ACTTTCGTTCTTGAT[CT][AG]A-3′ ) primers (Stoeck et al. 2010). Duplicate PCR reactions were performed in 50 μL volumes of: 1X Phusion High-Fidelity DNA buffer, 1 unit of Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA, #M0530S), 200 μM of dNTPs, 0.5 μM of each V4 forward and reverse primer, 3% DMSO, 50 mM of MgCl and 5 ng of either DNA or cDNA template per reaction. The PCR thermal cycler program consisted of a 98◦C denaturation step for 30 s, followed by 10 cycles of 10 s at 98◦C, 30 s at 53◦C and 30 s at 72◦C, and then 15 cycles of 10 s at 98◦C, 30 s at 48◦C and 30 s at 72◦C, and a final elongation step at 72◦C for 10 min, as described in Rodrı ́guez-Martı ́nez et al. (2012). PCR products were purified (Qiagen, #28104) and duplicate samples were pooled. The ∼400 bp cDNA and DNA PCR products were quality checked on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).

Sampling Locations:
SPOT (33◦ 33′ N, 118◦ 24′ W) - surface, DCM, 150 m, and 890 m
Port of LA (33◦ 42.75′ N, 118◦ 15.55′ W) - surface
Catalina (33◦ 27.17′ N, 118◦ 28.51′ W)-surface

For protocols see:
https://www.protocols.io/view/sample-collection-from-the-field-for-downstream-mo-hisb4ee
https://www.protocols.io/view/rna-and-optional-dna-extraction-from-environmental-hk3b4yn
https://www.protocols.io/view/18s-v4-tag-sequencing-pcr-amplification-and-librar-hdmb246

Nucleotide bases with a Q score lower than 20 for the last 30 bp of each sequence were trimmed. Paired-end sequences were merged using FLASh (Magoc and Salzberg 2011) with a minimum of 10 bp and maximum of 150 bp overlap between each sequence pair. Sequences shorter than 350 bp, longer than 460 bp, or which had an average quality score lower than 25 were discarded using QIIME v1.8 (Caporaso et al. 2010). Chimeric sequences were identified and removed, by either de novo or reference-based chimera checking (identify chimeric seqs.py in QIIME, intersection method). 

The code release v2 associated with this version of the dataset can be downloaded as a .zip file from the Supplemental Documents section of this page. Future code updates will be accessible from the GitHub repository https://github.com/shu251/V4_tagsequencing_18Sdiversity_q1.

BCO-DMO Data Manager Processing Notes:
* File "SRA_metadata_finalSHU.xlsx" imported into the BCO-DMO data system.
* Added SRA_run_ids from file "Listof_SRA_IDs.xlsx."
* added a conventional header with dataset name, PI name, version date
* modified parameter names to conform with BCO-DMO naming conventions
* blank values in this dataset are displayed as "nd" for "no data."  nd is the default missing data identifier in the BCO-DMO system.
* Split lat_lon column into "lat" and "lon" in decimal degrees.
* Added active html links to dataset for SRA Run IDs
*  For curatorial purposes and to satisfy the OCE Sample and Data Policy requirements, the contributor's github was forked to BCO-DMO's github, a release was made, and the .zip file containing that release downloaded to BCO-DMO's data servers. 
   * BCODMO/V4_tagsequencing_18Sdiversity_q1 forked from shu251/V4_tagsequencing_18Sdiversity_q1
   * Release v2 created: https://github.com/BCODMO/V4_tagsequencing_18Sdiversity_q1/tree/v2

Planktonic marine microbial communities consist of a diverse collection of bacteria, archaea, viruses, protists (phytoplankton and protozoa) and small animals (metazoan). Collectively, these species are responsible for virtually all marine pelagic primary production where they form the basis of food webs and carry out a large fraction of respiratory processes. Microbial interactions include the traditional role of predation, but recent research recognizes the importance of parasitism, symbiosis and viral infection. Characterizing the response of pelagic microbial communities and processes to environmental influences is fundamental to understanding and modeling carbon flow and energy utilization in the ocean, but very few studies have attempted to study all of these assemblages in the same study. This project is comprised of long-term (monthly) and short-term (daily) sampling at the San Pedro Ocean Time-series (SPOT) site. Analysis of the resulting datasets investigates co-occurrence patterns of microbial taxa (e.g. protist-virus and protist-prokaryote interactions, both positive and negative) indicating which species consistently co-occur and potentially interact, followed by examination gene expression to help define the underlying mechanisms. This study augments 20 years of baseline studies of microbial abundance, diversity, rates at the site, and will enable detection of low-frequency changes in composition and potential ecological interactions among microbes, and their responses to changing environmental forcing factors. These responses have important consequences for higher trophic levels and ocean-atmosphere feedbacks. The broader impacts of this project include training graduate and undergraduate students, providing local high school student with summer lab experiences, and PI presentations at local K-12 schools, museums, aquaria and informal learning centers in the region. Additionally, the PIs advise at the local, county and state level regarding coastal marine water quality.

This research project is unique in that it is a holistic study (including all microbes from viruses to small metazoa) of microbial species diversity and ecological activities, carried out at the SPOT site off the coast of southern California. In studying all microbes simultaneously, this work aims to identify important ecological interactions among microbial species, and identify the basis(es) for those interactions. This research involves (1) extensive analyses of prokaryote (archaean and bacterial) and eukaryote (protistan and micro-metazoan) diversity via the sequencing of marker genes, (2) studies of whole-community gene expression by eukaryotes and prokaryotes in order to identify key functional characteristics of microorganismal groups and the detection of active viral infections, and (3) metagenomic analysis of viruses and bacteria to aid interpretation of transcriptomic analyses using genome-encoded information. The project includes exploratory metatranscriptomic analysis of poorly-understood aphotic and hypoxic-zone protists, to examine their stratification, functions and hypothesized prokaryotic symbioses.