Emp Sample Submission Guide Pfqdjmw
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EMP Sample Submission Guide Luke Thompson,Gail Ackermann,Greg Humphrey,Jack Gilbert,Janet Jansson,Rob Knight Abstract This protocol was designed for collaborators with the Earth Microbiome Project to contribute samples in a standardized fashion. Raw, frozen samples are submitted in individually labeled tubes, 10 aliquots (identical replicates) per sample. Please note that unsolicited samples cannot be accepted. Citation: Luke Thompson,Gail Ackermann,Greg Humphrey,Jack Gilbert,Janet Jansson,Rob Knight EMP Sample Submission Guide. protocols.io dx.doi.org/10.17504/protocols.io.pfqdjmw Published: 13 Apr 2018 Guidelines Samples for this study will ideally meet the following criteria: Fresh or frozen sample (no preservatives) in ~10 aliquots Metadata well characterized Large proportion of unmapped diversity in at least some samples Non-human-associated and non-human-perturbed samples will be given priority Shipping Guidelines For non-soil samples only (e.g., feces, sediments, biofilms, organic material, water filters), no special permits should be necessary. However, material transfer agreements (MTAs) may be required, and these should be negotiated between the sender and recipiment as early as possible. 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. Samples should be shipped with FedEx overnight (or DHL for international shipments). Soil samples must be shipped to the permit holder. 1 Published: 13 Apr 2018 This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited MTAs may also be required by the sending and receiving institutions, and these should be agreed upon as early as possible. Materials BD SWUBE™ collection and transport system 281130 by Bd Millipore Express PLUS Membrane Filter, polyethersulfone, Hydrophilic, 0.22 µm, 47 mm GPWP04700 by Millipore JGWP04700 | Omnipore Membrane Filter, PTFE, Hydrophilic JGWP04700 by Millipore Screw Cap Micro Tube, 2 ml, PP, with skirted base, with knurls, with assembled cap, no print, sterile, 100 pcs./bag 72.694.005 by Sarstedt Protocol Sampling Step 1. 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 Step 2. Please choose the one of the following sample types. Procedures specific to sample types Step 3 - Bulk unaltered (Examples: soil, sediment, feces). Split fresh (or frozen) sample into 10 2-mL screw-cap bead beater tubes, ideally with at least 200 mg biomass, flash freeze in liquid nitrogen (if possible), and store at -80 °C (or -20 °C). TEMPERATURE -80 °C Additional info: Storage REAGENTS Screw Cap Micro Tube, 2 ml, PP, with skirted base, with knurls, with assembled cap, no print, sterile, 100 pcs./bag 72.694.005 by Sarstedt Labeling Step 4 - Bulk unaltered (Examples: soil, sediment, feces). 2 Published: 13 Apr 2018 This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited To track the provenance of these aliquots, we employ the following QR barcoding scheme: Tubes should be 2-mL screw-cap bead beater tubes to enable direct use in DNA extraction. Labels should be affixed to aliquot tubes before shipping. QR codes have the format doe.99.s003.a05, where "doe" is the PI name, "99" is the study ID, "s003" is the sample number, and "a05" is the aliquot number. QR codes (version 2, 25x25) are printed on Cryogenic Direct Thermal Labels, 1.125" x 0.75" rectangluar labels and 0.437" circular cap labels (GA International, part no. DFP-70) using a Zebra model GK420d printer and ZebraDesigner Pro software for Windows. Before aliquots are put away, QR codes are scanned into a sample inventory spreadsheet using a QR scanner. Procedures specific to sample types Step 3 - Bulk fractionated (Examples: sponges, corals, turbid water). Fractionate the sample as appropriate for your sample type. Split into 10 2-mL screw-cap bead beater tubes, ideally with at least 200 mg biomass, flash freeze in liquid nitrogen (if possible), and store at -80 °C (or -20 °C). TEMPERATURE -80 °C Additional info: Storage REAGENTS Screw Cap Micro Tube, 2 ml, PP, with skirted base, with knurls, with assembled cap, no print, sterile, 100 pcs./bag 72.694.005 by Sarstedt Labeling Step 4 - Bulk fractionated (Examples: sponges, corals, turbid water). 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 in Guidelines). 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. Procedures specific to sample types Step 3 - Swabs (Examples: biofilms, feces). Take 10 replicate swabs using 5 BD SWUBETM dual cotton swabs with wooden stick and screw cap. Place cap on swabs and store in -80 °C (or -20 °C). TEMPERATURE 3 Published: 13 Apr 2018 This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited -80 °C Additional info: Storage REAGENTS BD SWUBE™ collection and transport system 281130 by Bd Labeling Step 4 - Swabs (Examples: biofilms, feces). 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 in Guidelines). 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. Procedures specific to sample types Step 3 - Filters (Example: 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 2-mL screw-cap bead beater tubes, flash freeze in liquid nitrogen (if possible), and store at -80 °C (or -20 °C). REAGENTS Millipore Express PLUS Membrane Filter, polyethersulfone, Hydrophilic, 0.22 µm, 47 mm GPWP04700 by Millipore JGWP04700 | Omnipore Membrane Filter, PTFE, Hydrophilic JGWP04700 by Millipore Screw Cap Micro Tube, 2 ml, PP, with skirted base, with knurls, with assembled cap, no print, sterile, 100 pcs./bag 72.694.005 by Sarstedt Labeling Step 4 - Filters (Example: water). To track the provenance of these aliquots, we employ the following QR barcoding scheme: Tubes should be 2-mL screw-cap bead beater tubes to enable direct use in DNA extraction. Labels should be affixed to aliquot tubes before shipping. QR codes have the format doe.99.s003.a05, where "doe" is the PI name, "99" is the study ID, "s003" is the sample number, and "a05" is the aliquot number. QR codes (version 2, 25x25) are printed on Cryogenic Direct Thermal Labels, 1.125" x 0.75" rectangluar labels and 0.437" circular cap labels (GA International, part no. DFP-70) using a Zebra model GK420d printer and ZebraDesigner Pro software for Windows. Before aliquots are put away, QR codes are scanned into a sample inventory spreadsheet 4 Published: 13 Apr 2018 This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited using a QR scanner. Procedures specific to sample types Step 3. Place filters in micro centrifuge tubes or cry-vials, flash freeze in liquid nitrogen (if possible), and store at -80 °C (or -20 °C). TEMPERATURE -80 °C Additional info: Storage Warnings Please refer to the SDS (Safety Data Sheet) for safety and hazard information. 5 Published: 13 Apr 2018 This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
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