Illumina amplicon seq of eDNA sample using 12SV5 primers
Metabarcoding library preparation followed a two-PCR protocol, with target fragment amplification being conducted in the first-step PCR and index and sequencing adapter incorporation in the second-step PCR. Two different primer sets were used in parallel for amplification of two different fragments of the mitochondrial 12S rRNA fragment: 1. The MiMammal primer set (Ushio et al. 2017) and the 12SV5 primer set (Riaz et al. 2011). Primers for first-step PCRs consisted of the PCR primer sequences of the original publications for target fragment amplification, preceded by either a 5  7 bp (MiMammal primers) or 6 bp (12SV5 primers)-long fully degenerate nucleotide sequence on the 5 end, preceded the TruSeq Sequencing primer sequence. First-step PCRs were conducted in triplicates for each sample, which were then pooled after verification of PCR success. Each PCR reaction consisted of 5 l template (eDNA sample, mock community or nuclease-free water), 0.25 l Q5 High-Fidelity Polymerase (2,000 U/ml, New England Biolabs), 5 l Q5 Reaction Buffer, 0.2 mM of dNTPs, 0.3 M of forward and reverse primer, respectively and nuclease-free water up to final reaction volume of 25 l. Cycling conditions were as follows: Initial denaturation at 98C for 30 sec, followed by 35 cycles of denaturation at 98C for 20 sec, annealing at 62.5C (MiMammal) or 57C (12SV5) for 30 sec, extension at 72C for 60 sec and a final extension at 72C for 7 min. Due to co-amplification of non-target fragments from algae and bacteria, resulting in double-bands using MiMammal primers, PCR products of MiMammal PCRs were excised from a 2% agarose gel and subsequently cleaned using the Wizard SV Gel and PCR Clean-Up System (Promega). 12SV5 PCR products were checked for PCR success on a 2% Agarose Gel and subsequently cleaned using SPRISelect magnetic beads with the Left Side Selection protocol and a beads to sample ratio of 1.2. Cleaned PCR Products were then subjected to second-step PCR for incorporation of Nextera indices and illumina Sequencing adapters. PCR reactions consisted of 5 l template DNA, 0.2 l Q5 High-Fidelity Polymerase (2,000 U/ml, New England Biolabs), 4 l Q5 Reaction Buffer, 0.2 mM of dNTPs, 0.5 M of P5/7 forward and reverse primer, respectively (Table 1) and nuclease-free water up to final reaction volume of 20 l. Cycling conditions were as follows: Initial denaturation at 98C for 30 sec, followed by 15 cycles of denaturation at 98C for 10 sec, annealing at 66C for 30 sec, extension at 72C for 30 sec and a final extension at 72C for 2 min. PCR success was verified via 2% Gel Electrophoresis and PCR Products were cleaned using the SPRISelect beads as described above. Cleaned PCR products were pooled (using 5 l of each individual PCR product) and final library and concentrated using the DNA Clean & Concentrator-5 (Zymo Research) to a final total volume of 30 l. Library concentration was quantified with the NEBNext Library Quant Kit for Illumina (New England Biolabs).