Step 1. Sampling

Samples should be collected fresh and split into 10 aliquots and then frozen, or collected and frozen and subsequently split into 10 aliquots with minimal perturbation. Do not use any buffers or solutions to preserve your samples. Do not use RNAlater! Ethanol (50-95%) is acceptable. Aliquot size should be sufficient to yield 10-100 ng genomic DNA, which is approximately 107 to 108 cells. For low-biomass samples, such as certain water samples and biofilms, please contact us.

Procedures specific to sample types are as follows:

  • Bulk unaltered (e.g. soil, sediment, feces) — Split fresh (or frozen) sample into 10 microcentrifuge tubes or cry-vials, ideally with at least 200 mg biomass, flash freeze in liquid nitrogen (if possible), and store at -80 °C (or -20 °C).
  • Bulk fractionated (e.g. sponges, corals, turbid water) — Fractionate the sample as appropriate for your sample type. Split into 10 micro centrifuge tubes or cry-vials, ideally with at least 200 mg biomass, flash freeze in liquid nitrogen (if possible), and store at -80 °C (or -20 °C).
  • Swabs (e.g. biofilms) — Take 10 replicate swabs using 5 BD SWUBE dual cotton swabs with wooden stick and screw cap (cat. no. 281130). Cap swabs and place in -80 °C (or -20 °C).
  • Filters (e.g. water) — Filter water through 10 replicate filters: 47 mm diameter, 0.2 um pore size, polyethersulfone (preferred) or hydrophilic PTFE filters. Catalog numbers from Millipore: GPWP04700 (polyethersulfone), JGWP04700 (hydrophilic PTFE “Teflon”). Place filters in micro centrifuge tubes or cry-vials, flash freeze in liquid nitrogen (if possible), and store at -80 °C (or -20 °C).

Step 2. Labeling

Label your tubes clearly in permanent marker with a descriptive name. For example, use the PI or contact person’s last name, the study number (provided by the Knight lab), the sample type, and sequential number: “Jones99.soil.1”. This is the default sample numbering system used in the metadata template (see below). Other naming conventions are acceptable, but please make sure the names match those in the metadata CSV file. Please do not number the aliquots; you should submit 10 identically labeled aliquots for each sample.

Step 3. Metadata

High-quality environmental metadata is essential for meaningful analysis and is required for all samples before they are processed. Please go to the EMP Metadata Guide and read the general instructions for fields required by Qiita (database for EMP data), MIMS (Minimal Information about a Metagenomic Sequence), and your specific environmental package (sample type). For your environmental package (or more if you have multiple sample types), download the metadata template CSV file and README file, fill in the metadata template, and email it to Luke Thompson and Gail Ackermann. If you are sending additional samples for possible later processing (so-called “Tier 2″ samples as discussed with the EMP coordinators), please send a separate metadata file for these.

Step 4. Shipping

For all samples please follow these instructions:

  1. Samples in tubes of appropriate size should be shipped on crushed dry ice in a styrofoam container. Please ensure there is enough dry ice to survive for the duration of the trip.
  2. Place samples in dry ice. If there is space between the top of the ice and the lid, fill with paper, or other packing materials, to secure the samples during transit.
  3. Tape styrofoam container to secure the lid.
  4. Tape a list of samples to the outside of the styrofoam container for sample identification. Sample names should match the sample names in your metadata file.
  5. If you are sending additional samples for possible later processing (so-called “Tier 2” samples as discussed with the EMP coordinators), please group these separately and mark as “Tier 2”, with a separate list of samples.
  6. Place styrofoam container into larger cardboard shipping box. Pack the box as needed with paper or other packing material as needed to fill up the empty space in the box and secure the styrofoam box during shipping.
  7. Seal cardboard box with tape.
  8. Ship samples using FedEx overnight (or DHL for international shipments). Ship Monday through Wednesday to avoid samples that are delayed arriving on a weekend.

For non-soil samples only (e.g., feces, sediments, biofilms, organic material, water filters):

  • Ship to the following address and send tracking number to Greg Humphrey.
    University of California, San Diego
    Knight Lab / Attn: Greg Humphrey
    BRF II Room 1220D
    9500 Gilman Drive, MC 0763
    La Jolla, CA 92093-0763
    Phone: 858-246-1964

For soil samples only (sediments are not considered soils):

  • Regulated domestic soils must be shipped with the Permit to Receive Soil issued through the Animal and Plant Health Inspection Service of the United States Department of Agriculture. Collaborators should know whether or not their soils are regulated. If domestic soils are not regulated, the permit is not needed.
  • The PPQ Form 550 Black/White label (sticker) must be affixed to the outside of the shipment for any soils shipping from outside the USA, Guam, Hawaii, Puerto Rico, or the US Virgin Islands.
  • If you have a mixture of soil and non-soil samples, you may ship them all together to PNNL.
  • Samples should be shipped with FedEx overnight (or DHL for international shipments). Soil samples must be shipped to PNNL and must be addressed to Janet Jansson as she is the permit holder. Ship to the following address and send tracking number to Eric Bottos.
    Janet Jansson / Attn: Eric Bottos (BSF/2245, no log#)
    PNNL for the US DOE
    790 6th Street
    Richland, WA 99354
    Phone: 509-375-2440

Step 5. Data release

DNA sequence data will be submitted to the European Nucleotide Archive as raw FASTQ files shortly after generation, as required by the funding agencies. Metabolomic data will be submitted to appropriate repositories on a similar timeline. Collaborators will be notified when their data are ready, and are free to access and publish data from their samples.

Large meta-analyses of the results will be directed by a consortium of researchers at UC San Diego, Pacific Northwest National Lab, Argonne National Lab, and other institutions. If you are interested in helping with these analyses or have questions, please contact Dr. Luke Thompson.

If you have concerns with the sharing and release of data, please contact the EMP Steering Committee (Rob Knight, Janet Jansson, and Jack Gilbert).