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This protocol describes our sampling strategy and techniques for the NOAA-CalCOFI Ocean Genomics (NCOG) project. In summary, seawater for DNA and RNA from multiple depths is filtered onto Sterivex filters at each station. Although the protocol is specific to this project, it is easily adaptable for other field-based sampling.

18S and 16S amplicon library preparation protocol. DNA or RNA is usually extracted with our automated protocols from sterivex but other types of samples are occasionally used. If using RNA, first generate cDNA with Invitrogen's SuperScript III First-Strand Synthesis System. Blanks from the nucleic acid extraction should always be used as negative controls. Normally, DNA is diluted 10-fold after extraction and 1 μL of the diluted DNA or the cDNA is used for template in the PCR reaction. If DNA concentrations are very low, 1 μL of the undiluted DNA may need to be used. The library is generated with a 1-step PCR, i.e. amplicons are amplified and barcoded simultaneously. Amplicons can be generated for 16S (515F-Y/926R primers from Parada et al. 2015), 18Sv4 (V4F/V4RB primers from Balanzo et al. 2015), and 18Sv9 (1389F/1510R primers from Amaral-Zettler et al. 2009). Barcoded primers for all of these amplicons are attached to this protocol. Typically, the primers are pre-mixed into 96-well plates such that one set of 8 unique F primers (plate rows) and one set of 12 unique R primers (plate columns) are mixed to create 96 unique combinations. Reactions can then be run in a 96 well plate where template plate positions correspond to unique barcode plate positions. The i5 barcode is inline to generate higher sequence quality but also requires a customized demultiplexing workflow. When sequencing, only demultiplex based on the i7 index first. Scripts to then demultiplex based on the i5 index can be found on our Github here. We also highly recommend including mock communities in every 96-well plate.

A mostly automated protocol for extraction of total RNA from seawater filtered onto a Sterivex filter (Cat. No. SVGP0150). Reagents come from the Macherey-Nagel NucleoMag RNA kit (Cat. No. 744350). Automated liquid handling is performed on an eppendorf EpMotion 5075t with multi-channel pipettes. This protocol assumes that the Sterivex is sealed with tube sealant on the male end and a male-luer lock plug on the female end. Using this protocol, we routinely extract ~80 samples at a time. The protocol has been modified to increase to an 800 µL starting volume. This normally results in less Lysis Buffer MR1, Binding Buffer MR2, and Reducing Agent TCEP supplied than is needed. Extra reagents can and should be purchased separately. The kit manual can be found here: https://www.mn-net.com/media/pdf/23/c2/23/Instruction-NucleoMag-RNA.pdf Additional notes from the manufacturer on using the kit with the epMotion can be found here: https://www.mn-net.com/media/pdf/bd/7e/53/AN-NucleoMag-RNA-epMotion5075.pdf . Note that we have several additional modifications to their protocol. The epMotion program is attached here.

A mostly automated protocol for extraction of genomic DNA from seawater filtered onto a Sterivex filter (Cat. No. SVGP0150). Reagents come from the Macherey-Nagel NucleoMag Plant Kit for DNA purfication (Cat. No. 744400). Automated liquid handling is performed on an eppendorf EpMotion 5075t with multi-channel pipettes. This protocol assumes that the Sterivex is sealed with tube sealant on the male end and a male-luer lock plug on the female end. Using this protocol, we routinely extract ~80 samples at a time. The protocol has been modified to increase to an 800 µL starting volume. This normally results in less Lysis Buffer MC1 and Binding Buffer MC2 than is needed. Extra binding buffer should be purchased separately. The kit manual can be found here: https://www.mn-net.com/media/pdf/09/46/a0/Instruction-NucleoMag-Plant.pdf Additional notes from the manufacturer on using the kit with the epMotion can be found here: https://www.mn-net.com/media/pdf/95/2c/ef/AN-NucleoMag-Plant-epMotion5075.pdf . Note that we have modifications to their protocol. The epMotion program is attached here.