title: DNA metabarcoding of chironomid communities
design: Pooled chironomid samples were dried for at least 24 h at 60C, grinded and DNA was extracted from each sample with two technical replicates following a high salt DNA extraction protocol. A 421 bp COI fragment was amplified using the BF2/BR2 primer set (Elbrecht and Leese 2017) in a two-step PCR reaction (Elbrecht & Steinke 2018). The DNA concentration was quantified using a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit; Advanced Analytical, Ankeny, USA). The library was purified and size selected (retaining fragments of >300 bp) with left size selection of magnetic beads (SpriSelect, Beckmann Coulter, Bread, CA, USA, ratio: 0.76x). Purified PCR products were pooled into a library proportional to the number of specimens in each sample to ensure all specimens are sequenced with comparable sequencing depth. The library was sent for 2 x 250 bp Paired-end sequencing on a MiSeq Illumina system (v3) run (with 5% PhiX spike into increase sequence diversity).