DungBeetleGut_Library_27_16s
Mini-barcode sequences for the 12SrRNA and 16SrRNA mitochondrial (mtDNA) ribosomal genes were amplified using the primers  12SV5F and 12SV5R (Riaz et al., 2011) and 16Smam1 and 16Smam2 (Taylor 1996) to target vertebrate and mammal species, respectively. For the 12SV5F primer, the first nucleotide was changed to a degenerate base (5 - YAGAACAGGCTCCTCTAG - 3) to allow binding in more mammal species, as suggested by Kocher et al. (2017). The PCR protocols for both mini-barcodes followed Rodgers et al. (2017), with minor modifications: 1x buffer (TrisHCl 20 mM pH 8.4 and KCl 50 mM), 0.4 mM of each primer, 0.2 mM dNTP (Invitrogen), 4 and 2 mM MgCl2  for 16SrRNA and 12SrRNA, respectively, 1.5 U Platinum Taq polymerase (Invitrogen), and 3 l of template DNA. Cycling conditions were 10 min at 95C, followed by 42 cycles of 30 s at 95C, 30 s at 64C and 50C for 16S rRNA and 12S rRNA, respectively, and 1 min at 72C, with a final extension of 10 min at 72C. All PCRs were prepared in a dedicated room within a UV-sterilized hood and included a no-template DNA as negative control to check for contamination. PCR amplification success was checked in 1.5% agarose gel. For large-scale sequencing, the libraries were cleaned using magnetic beads (Agencourt AMPure XP  Beckman Coulter), quantified in a Qubit fluorometer (Thermo Fisher, Waltham, Massachusetts, USA), normalized to 50 ng/l, and indexed using a Nextera Index kit (Illumina, San Diego, California, USA). The paired-end metabarcoding sequencing was performed on an Illumina iSeq platform, using an iSeq 100 v2 300 Cycle Reagent kit (2x150 bp), for a total of 70,000  100,000 reads per each sequenced library.