Metabarcoding sequencing of a universal rbcL region using bird feces
Each sample is a de-multiplexed version of a pooled sample of DNA extracted from bird feces and amplified using a universal plant mini-barcode within the rbcLa subregion. For the mini-barcode amplification, the primers rbcL1 Forward  5 TTGGCAGCATTYCGAGTAACTCC 3 and rbcLB 5 AACCYTCTTCAAAAAGGTC 3 were used (LITTLE, 2014; PALMIERI et al., 2009). This mini-barcode results in a 184 bp fragment, which is therefore ideal for degraded plant DNA found in droppings (SHUTT et al., 2021). Six tagged versions of the primer pair were synthetized with an Illumina sequencing overhang adapter and one of six five-base pair tags obtained from Axtner et al. 2019. Each sample name consists of abbreviated genus_species name_sample ID_Pool number_Tag. The sample ID is the same as the capture number of the individual. The Pool number indicates which Pool the sample was sequenced in, with each pool containing six samples marked by unique Tags. The Tag letter corresponds to the Tags described in Axnter et al. 2019. Following amplification, PCR products were visualized using gel electrophoresis in a 1.5% agarose gel. To increase yield and guarantee successful amplification, each sample was amplified three times and 6 L of each PCR product was combined into a single sample of 18 L. Samples were pooled according to similar product concentration (ng/ L) estimated by visual inspection of band strength in the electrophoresis gel. When possible, PCR products from birds of the same species were grouped together. We avoided pooling samples that had strong bands with weakly amplified samples. A volume of 5 L was taken from each of the six samples and combined in a single tube, resulting in a final volume of 30 L. A grouped sample of blanks was also combined using 5 L of each tag associated blank, resulting in a final volume of 30 L.