Table of Contents

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

DNA and cDNA (RNA) V4 tag sequences that characterize the spatiotemporal (monthly and seasonal) variability of eukaryotic microbial assemblages in the upper euphotic zone of the eastern North Pacific at 5m depth and Deep chlorophyll maximum (DCM) from 2003 - 2018.

These genetic sequences under BioProject PRJNA311248 will be made available at National Center for Biotechnology Information (NCBI) on June 1st, 2020.

Methodology:

Seawater samples were collected from SPOT station (3333N, 11824W) from two depths. The SPOT station was sampled from 5m and DCM using 10 L niskin bottles mounted on a CTD rosette. The depth of the DCM was determined by in situ real-time fluorescence data from the CTD during each sample collection. Seawater samples were pre-screened through a 200 um nitex mesh and 80 um nitex using gravity filtration and and in-line filtration. Seawater collected at 5m (2L) and DCM (2L) were filtered onto a GF/F filter (nominal pore size 0.7 um; Whatman) and filters were stored in Caron Lab lysis buffer for DNA extraction and stored dry for RNA extraction. Filters were flash frozen in liquid nitrogen and stored in -80 C until further processing.
Total DNA extraction was accomplished by modifying the protocol described by DNeasy Plant Mini Kit (Qiagen, #69104). Filters were thawed for 5 min and 65C in a heat block and bead beaten by adding RNase-free silica beads and vortexed for 5 min with the addition of RNase A. Lysate was syringe-filtered and the appropriate proportions of neutralization buffer was added to the lysate. Total DNA was then extracted using DNeasy Plant Mini Kit, as per manufacturers instructions.
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, #74104), as per manufacturers instructions. Filters were thawed on ice and 1.5 mls of RLT + (beta-Mercaptoethanol) was added to each filter and bead beaten by adding RNase-free silica beads and vortexed for 5 min. Genomic DNA was removed during RNA extraction (Qiagen, #79254) and checked for residual genomic DNA by performing PCR using (Stoeck et al., 2010) primers and observing an absence of amplification in an agarose gel. RNA was reverse transcribed into cDNA using iScript Select cDNA Synthesis Kit (BioRad, #1708896) with Random Primers. The resulting DNA and cDNA from each sample were PCR amplified using (Stoeck et al., 2010) primers with attached Illumina Adaptors (Forward- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CASCYGCGGTAATTCC, Reverse- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-ACTTTCGTTCTTGATYRA). For each sample, we added 10 ng of genetic material to PCR tubes containing 0.5 um of each forward and reverse primer and 1X Q5 High Fidelity Master Mix for a final PCR volume of 25 ul and final concentration reaction mixture of .4 ng/ul. The PCR thermal profile had an initial activation step (Q5 specific) of 98C for 2 minutes, followed with 10 cycles of 98C for 10 seconds, 53C for 30 seconds, 72C for 30 seconds, and 15 cycles of 98C for 10 seconds, 48C for 30 seconds, and 72C for 30 seconds, and a final extension of 72C for 2 minutes. PCR products were purified using an AMPure bead clean up (Beckman Coulter #A63881). After purification, PCR products were normalized to one another and indexed using Illumina-specific P5 and P7 indices. Final indexes samples were pooled at equimolar concentration of 10 uM and sequenced using 250 bp PE sequences (Laragen).

The CTD was operated per Sea-Birds suggested methods, and was powered up and allowed to stabilize at approximately 0-5 meters prior to profiling. The instrument was lowered at 40 m per minute (maximum rate) for the entire depth of the sampling site (0-890 m).

Sampling ranges:
SPOT DNA samples were extracted from 2003-2018 (omitting 2017). Ten DNA samples were extracted for each month for two depths (5 m and DCM) from 2003-2018 (omitting 2017).
SPOT RNA samples were extracted from SPOT cruises from 2013 - 2016. Three years of RNA samples were collected for each month for two depths (5m and DCM) from 2013 - 2016. RNA samples were extracted for each month consecutively from 2013 - 2016; full dataset from 2013-2016 for RNA samples.

Location:
The SPOT station (33˚33 N, 118˚24 W) is located approximately 10 miles offshore from the Port of Los Angeles in the southern California Bight.

Sequences have been analyzed using QIIME2 - DADA2 software (qiime2-2018.8).

The following programs were used to collect and process CTD data: SEASAVE V.7.2 (to display, record and playback data), SEACON (to enter calibration coefficients and configuration records) and Sea-Bird software Seasoft (data is recorded at a full scan rate at 24 Hz).

BCO-DMO Data Manager Processing Notes:
* added a conventional header with dataset name, PI name, version date
* modified parameter names to conform with BCO-DMO naming conventions. Removed spaces and replaced with underscores.
* added Latitude and Longitude columns with values in decimal degrees from a "Coordinate" column containing locations as strings with decimal degrees and directionals.
* Replaced comma in geographic locations column with a semicolon to better support export as csv format.
* Removed empty columns named: filename3, filename4, assembly, fasta_file

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.