title: SamplingPoint-P8_Tag-ATTCC_MiniBarcode-12S
design: Mini-barcode sequences for the 12SrRNA and 16SrRNA mitochondrial (mtDNA) ribosomal genes were amplified using primers previously described to target vertebrate (12SV5F and 12SV5R; Riaz et al., 2011) and mammal (16Smam1 and 16Smam2; Taylor 1996) 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 mini-barcode primers were designed to amplify approximately 135-139 bp (12SrRNA) and 130-134 bp (16SrRNA). Unique identifiers (tags), described by Axtner et al. (2019), were added to both forward (F) and reverse (R) primers to mark each insect bulk (Supplementary Table S1), reducing the sequencing cost. PCR protocols followed Rodgers et al. (2017) and were carried out within an UV-sterilized hood in an iDNA-dedicated PCR room. To check for contamination, PCR amplifications included a non-template sample as negative control. The amplified products were visualized on 1.5% agarose gels by electrophoresis. For large-scale sequencing, we pooled the samples in four sets of sequencing samples (two for 12SrRNA and two for 16SrRNA sequences). The sets of sequencing samples 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 reads per each of the four sets of sequencing samples.