“In silico” PCR
We then went on to check by “in silico” PCR the new mini-barcodes (Table 1) and two eukaryotic mini-barcodes of the 28S rRNA gene, as the mini-barcodes most widely used in next generation sequencing (NGS) diet studies (Pompanon et al., 2012): nucLSUDf1/nucLSUDr1 (Sonnenbe et al., 2007) and Short28SF/Short28SR (Vestheim & Jarman, 2008). In addition, we compared the taxonomic coverage and resolution of the mini-barcodes to those of a reference eukaryotic primer set F566/R1200 (Hadziavdic et al., 2014) (Table 1). This F566/R1200 barcode has been identified as the best eukaryotic barcode among ”de novo” designed and previously existing universal eukaryotic 18S rRNA barcodes (Hadziavdic et al., 2014).
All primers were “in silico” tested using the program ecoPCR (Ficetola et al., 2010) and Obitools (Boyer et al., 2016). First we used theobisilva function to download the ecoPCR format SILVA 132 SSU reference (cleaned to keep only high quality sequences) and non-redundant dataset. Likewise, we downloaded the ecoPCR format SILVA 132 LSU reference dataset. For large subunits (LSU), SILVA has no non-redundant version. Second, we used the function ecotaxstat of ecoPCR (Ficetola et al., 2010) to test the taxonomic coverage of the 3 designed mini-barcodes, F566/R1200 eukaryotic long barcode (650bp), and nucLSUDf1/nucLSUDr1 and Short28SF/Short28SR 28S mini-barcodes on Bacteria, Archaea and Eukarya. In the same way, we used the functionsecotaxstat and ecotaxspecificity to test their taxonomic coverage and resolution capacity on 10 eukaryotic phyla (Supplemental information Table S3): Apicomplexa, Nematoda and Platyhelminthes including main parasites of birds; Phragmoplastophyta, Annelida, Arthropoda and Mollusca which may appear in birds’ diets; Ascomycota and Basidiomycota to check for fungal sample contamination; and Vertebrata to check if the barcodes could identify the host. Figure 3 focuses on the phyla Phragmoplastophyta, Apicomplexa, Arthropoda, Nematoda and Platyhelminthes, as we anticipated these would be the taxa mostly represented in the diet and parasites of birds. The ecotaxspecificity option -e 0 was used, which does not allow any mismatches. Taxon classification was according to Silva’s reference database (www.arb-silva.de).