title: COI  metabarcoding: freshwater benthos
design: Freshwater  benthos samples were homogenized in a blender, slurry added to a tube, spun  down, and excess ethanol poured out.  Residual ethanol was evaporated.  Homogenate subsampled into 2ml lysing tubes and homogenized in a  FastPrep-24 tissue homogenizer.  Samples were treated with Proteinase K overnight, extracted with a  NucleoSpin Tissue Kit (Machery-Nagel) according to manufacturer's protocol  with a final elution in 30ul molecular grade water. Two COI amplicons (BR5 and F230R) were  amplified. First PCR used standard  primers. Second PCR used standard  primers with Illumnina adapters. PCR  products were checked on a gel, purified using MinElute DNA purification  system, and quantified using a Quant-iT PicoGreen dsDNA assay. For each sample, equimolar amounds of each  amplicon were pooled and tagged using Nextera XT Index Kit. Indexed samples were pooled, purified using  magnetic bead purification, quantified using PicoGreen dsDNA assay, average  length determined on Agilent Bioanalyzer 2100 before sequencing on an  Illumina MiSeq using the V3 sequencing chemistry kit (2 x 300). 10% spike in of PhiX used as a control.