DNA-Seq of environmental sample: water
Laboratory procedures: DNA extraction: We cut each filter in halfone half was archived, while DNA from the other was extracted using the OMEGA E.Z.N.A Tissue DNA Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) following the manufacturers instructions but with an overnight incubation at 55C during the lysis step and two 100 L elutions in the final step. PCR, library preparation and sequencing: We quantified each sample using a Qubit 1 dsDNA HS Assay kit and subsequently prepared sequencing libraries using a 2-step PCR workflow as described in Timmers et al. (2024). We initially amplified DNA in triplicate 15 L reactions using targeted primers with Illumina overhangs (metabarcoding PCR), followed by a second round of amplification binding Illumina sequencing adapters and the i5/i7 Nextera XT indices to the amplified template in 25 L reactions to distinguish between multiplexed samples (indexing PCR). Metabarcoding PCR reaction components varied according to targeted primer (see Tables S3-S6) but were generally composed of 7.5 L of Q5 High-Fidelity 2 Master Mix (New England Biolabs, Ipswich, MA, USA), 0.250.75 L each of forward and reverse primer (at 10 M), 0.110.55 L Recombinant Albumin (NEB B9200S, 20 mg/mL), 4.85.89 L nuclease-free water (VWR, Radnor, PA, USA), and 1.0 L template DNA). We also prepared negative (no template) PCR controls, DNA extraction blanks, and field negatives for sequencing in the same manner. Thermocycler conditions varied based on targeted primer and are detailed in Tables S7S10. We verified amplification success on a 1.5% agarose gel. We pooled triplicate reactions prior to bead cleaning using the Mag-Bind TotalPure NGS kit (Omega Bio-Tek) in a ratio of 1.8 beads to sample following the manufacturers instructions and quantified using the Qubit 1 dsDNA HS Assay kit to determine yield. We carried out indexing PCR in 25 L reactions containing 12.5 L Q5 High Fidelity 2 Master Mix (NEB), 5 L of nuclease-free water (VWR), 2.5 L template DNA, and 2.5 L each of 1 M forward i5 and reverse i7 indexing primers. Indexing PCR conditions were as follows: 95C for 3 minutes, followed by 8 cycles of 95C for 30s, 55C for 30s, and 65C for 30s, with a final extension of 65C for 5 minutes. We verified amplification success on a 1.5% agarose gel by visualizing size increases. We purified each sample using magnetic beads as described above, quantified them using the Qubit 1 dsDNA HS Assay kit, and pooled them at equimolar concentrations. We checked pooled libraries for quality and length using an Agilent Technologies (Santa Clara, CA, USA) 2100 Bioanalyzer. Libraries were sequenced on an Illumina (San Diego, CA, USA) MiSeq platform using V3 chemistry (2 x 300 bp paired ends) at the UH Manoa Advanced Studies in Genomics, Proteomics and Bioinformatics (ASGPB) facility, targeting a depth of 100,000 reads per sample.
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