12S rRNA metabarcoding for Mock Community
PCR reactions consisted of 2.4 L 1x Buffer, 0.24 L dNTPs (0.20nM), 0.24 L of each primer at 0.20 nM, 0.48 L Human blocking primer (4.0 nM), 1.20 L BSA (0.02ng/L), 0.20 L Taq Phusion (0.40 ng/L), and 0.5 L of DNA. Thermocycling was conducted with the following parameters: initial denaturation at 98C for 3 minutes, followed by 40 cycles of 20 seconds at 98C, 20 seconds at the annealing temperature of 61C, and 20 seconds at 72C, with a final extension of 5 minutes at 72C. Amplicons were visualized using agarose gel (1.5% w/v) electrophoresis to verify amplification of DNA markers and to assess negative controls generated during PCR, extraction, filtration, and field collection. A second PCR was performed to incorporate dual molecular identifiers (MID) to individual samples on the same flow cell, allowing identification of the samples once the DNA of different samples are joined together for sequencing. Second PCR amplifications contained 1x PCR Buffer (5X Buffer Thermo), 0.2 mM dNTPs, 0.2 M F and R primers, 1.20 L BSA (0.02ng/L), 0.2 U Phusion (Thermo), and 0.8 L eDNA, in reactions of 12 L. The thermocycling conditions for the second PCR were initiated for 3 minutes at 98C, followed by 15 cycles of 98C for 30 seconds, 65C for 30 seconds, and 72C for 45 seconds, with a final extension at 72C for 3 minutes. Two PCR replicates were performed for each PCR 1 replicate. Following the second PCR, amplicon replicates of the same site and date were pooled together, and negative controls (field, extraction, and PCR) were pooled together.  For DNA purification, we used a magnetic bead protocol (AMPure XP cleanups) optimized for small DNA fragments. Two PCR 2 replicates were combined, after which magnetic beads at room temperature were added at a ratio of 1.8L per L of samples. Samples were homogenized by pipetting and then incubated for 510 minutes to allow DNA binding. The mixture was then placed on a magnetic plate, enabling bead aggregation and separation from the supernatant, which was discarded. Two washes were performed with 150L of freshly prepared 70% ethanol, followed by a 5-minute evaporation period to remove residual ethanol. Finally, the beads were resuspended in 20L of molecular-grade water, homogenized to release the bound DNA, and after a final magnetic separation, the supernatant containing purified DNA was transferred to a new tube. We then quantified the DNA concentration of each cleaned sample in order to merge them with the same equimolar quantities. For that, we used the DNA quantification kit Invitrogen Qubit Fluorometer. To prepare the Qubit working solution, the dsDNA HS Reagent was diluted 1:200 in dsDNA HS Buffer to yield a final volume of 200 L per samples (samples from the study + two standard from Qubit kit). For each standard samples, 190 L of working solution was dispensed, followed by the addition of 10 L of Qubit standard and a brief vortexing. For our study samples, the volume of the working solution was adjusted to 198 L so that, upon adding 2 L our study samples, the final volume reached 200 L. After all samples and standards were prepared, the tubes were incubated at room temperature for 2 minutes before proceeding with the instrument-specific reading procedure.