Metabarcoding of chironomidae: adult bulk samples
We followed a two-step PCR metabarcoding approach to amplify a 421 bp COI fragment. PCRs included negative controls and two technical replicates per sample. Primers were replaced by the corresponding fusion primers, including Illumina adapters for sequencing (P5 or P7) and inline barcodes of different length for individual tagging of samples. For each sample, the PCR product was pooled into a library proportionally to the number of chironomid specimens to ensure equal sequencing depth per specimen over all samples. Final libraries were purified and size-selected with magnetic beads, retaining mainly fragments >300 bp. Libraries were 2  250 bp (v2) pairedend sequenced on a MiSeq Illumina system.