title: DNA metabarcoding of Oreochromis mossambicus: gut content
design: We performed the amplification of two mitochondrial barcodes in separate PCR reactions. We targeted the 313 bp mitochondrial cytochrome c oxidase subunit I (COI) region with the primer pair of NexF_m1COIintF and NexR_jgHCO2198 (Leray et al., 2013) due to their suitability across a wide range of metazoan species. To identify other eukaryotic prey items, we used the 528F/706R primer set which amplifies a 350 bp region of the 18S ribosomal RNA gene (Ho et al., 2017 ). The blocking primer CCAAAGAATCAGAATAAGTGTTGSpacerC3 was implemented in the COI assay to minimize the amplification of host DNA. Overhangs allowing the second PCR step were added to all amplification primers. PCR reactions were prepared with a final volume of 10l containing 5l Multiplex PCR Master Mix (Qiagen, Hilden, Germany), 0.2l of each Primer, 2.8l of genomic DNA plus either 1.8l of primer blocking in the case of the COI PCR reactions or 1.8l of sterilized milliQ water in the case of the 18S PCR reactions. The PCR program followed an initial denaturation step at 95C for 15min, 30 cycles of denaturation at 95C for 0.5 min, annealing at 50C or 58C (for the COI and 18S primer pairs, respectively) for 1min and extension at 72C for 1min, and a nal extension step at 72C for 10 min. PCR blanks were included in each run and confirmed the absence of contamination. To minimize amplification biases, three PCR replicates were preformed per sample. The success of all amplification reactions was confirmed by electrophoresis. PCR products per sample were then pooled and used as template for the remaining library preparation steps as described in Silva et al. (2020) at the Centre for Molecular Analysis from CIBIO-InBIO as part of a service. The final libraries were sequenced in a MiSeq V2 2x250bp run with an estimated coverage of 5,000 reads per marker per sample.