“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).