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.