"Amplicon seq 16s & COI, lake water": Sample Greifensee
For each of the two metabarcodes (COI and 16S), PCR was performed once on each technical replicate for the lentic site only. PCRs on eDNA were carried out in 15µL volumes with final concentrations of 1x supplied buffer (Faststart TAQ, Roche, Inc., Basel, Switzerland), 1000 ng/µL BSA (New England Biolabs, Ipswich, MA, USA), 0.2 mMol dNTPs, 2.0 mMol MgCl2, 0.05 units per µL Taq DNA polymerase (Faststart TAQ, Roche, Inc., Basel, Switzerland), and 0.54 µMol of each forward and reverse prime. 2 µL of extracted eDNA was added that ranged in concentration from 0.05 to 0.55 ng/µL. This range was the outcome of DNA concentrations that were extracted following each of the six molecular workflows (Figure 2A). The thermal-cycling regime was 95 °C for 4 minutes, followed by 35 cycles of 95 °C for 30 seconds, either 55 °C or 48 °C (16S or COI respectively) for 30 seconds and 72 °C for 1 minute. A final extension of 72 °C for 7 minutes was carried out and the PCR was cooled to 10 °C until removed and stored at –20 °C until confirmation of products occurred. PCR products were confirmed by gel electrophoresis on a 1.4% agarose gel stained with ethidium bromide or GelRed (Biotium Inc., Hayward, CA USA). PCR products from the two genes (COI and 16S) for each technical replicate were pooled in equal proportions in a total of 20 µL resulting in 90 reactions that were then used as input DNA for library construction. Each of the 90 pooled reactions were then cleaned using AMPure XP beads following the recommended manufacture’s protocol, except 0.6 x reaction volume was used instead of 1.8 x based on recommended protocol for fragment size retention of >500 bp (p. 31, Nextera XT DNA 96 kit, Illumina, Inc., San Diego, CA, USA). We quantified each cleaned and pooled reaction using the Qubit (1.0) fluorometer following recommended protocols for the dsDNA BR Assay. The 90 pooled reactions were then each diluted with molecular grade DNA free water (Sigma-Aldrich, Co. LLC. St. Lewis, MO., USA) to 0.2 ng/µL following the recommended protocol for concentration of a DNA sample used for library construction (Nextera XT DNA 96 kit, Illumina, Inc., San Diego, CA, USA). This kit was used specifically because it requires only 1 ng of starting DNA compared with TrueSeq (Illumina, Inc., San Diego, CA, USA) which requires 1µg and was unrealistic to achieve from PCR on our technical replicates. Amplicons from the two metabarcode genes were pooled without any pre-labeling and were bioinformatically separated post-sequencing (see bioinformatics analysis section 2.3.1). Metabarcode libraries for the 90 technical replicates were prepared using the Nextera XT DNA 96 kit following manufacturer’s recommended protocols (Illumina, Inc., San Diego, CA, USA) except the final mixture of the denatured and pooled 90 libraries, plus the PhiX control, were diluted to 1:40 instead of 1:25. Paired-end (2x250nt) sequencing was performed on an Illumina MiSeq (MiSeq Reagent kit v2, 250 cycles) at the Genomic Diversity Center at the ETH, Zurich, Switzerland following manufacture’s run protocols (Illumina, Inc., San Diego, CA, USA). The MiSeq Control Software Version 2.2 including MiSeq Reporter 2.2 was used for the primary analysis and the de-multiplexing of the raw reads.