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To build a research platform for screening spider prey based on DNA metabarcoding of the ITS2 region, we designed PCR primers whose sequence matched nuclear 5.8S rRNA sequences of Hexapoda, but not those of Araneae. We first downloaded Hexapoda and Araneae sequences containing the 5.8S rRNA region from the NCBI Nucleotide database ( ) using the keywords described in Additional file 1. The 5.8S rRNA gene sequences were then extracted using the perl program available at -oldenburg.de/ibu/systematik-evolutionsbiologie/programme/, and then incomplete sequences lacking the core positions of the 5.8S rRNA region were removed from the data. The sequences of each order were subjected to multiple alignment using the program MAFFT v7.272  (Additional file 2).
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