title: Metabarcoding of benthos: freshwater kicknet samples
design: For test samples: Two paired benthos samples collected through kicknet sampling collected close to each other.  Ethanol or antifreeze preservative added and incubated 3 days at room temperature.  Samples homogenized.  For validation samples:  samples were stored in ethanol or antifreeze and stored at room temperature for 3 days prior to homogenization.   For half the samples the preservative was evaporated prior to DNA extraction (PRE). In POST samples, supernatant was just poured from the pellet without evaporating excess preservative.  Samples were extracted using a DNeasy PowerSoil kit with a final elution in 50ul buffer C6.  To verify purity of source preservatives, ethanol or antifreeze, DNA was extracted using a DNeasy PowerWater kit.  Three COI amplicons were amplified: BR5 (B/ArR5 primers), F230R (LCO1490/230_R primers), ml-jg (mICOIintF/jgHCO2198 primers) using a two-step PCR method.  First PCR used COI specific primers.  Second PCR used Illumina adapter-tailed primers.  Equimolar amounts of each amplicon was pooled before dual indexing with a Nextera XT DNA library preparation kit.  Illumina sequencing used a MiSeq V3 sequencing kit (300 x 2).  We sequenced negative control reactions (no DNA template) for antifreeze, ethanol, DNA extraction kits (with and without water added), and PCRs.