title: Metabarcoding of aquatic invertebrates with Nanopore sequencing Protocol A
design: PCR was prepared in 50l volumes with use of 25l of 2 Multiplex PCR Master Mix Plus (Qiagen, Hilden, Germany), 2l of 10pmol of each primer, 19l of nuclease-free molecular grade water (VWR Canada, Mississauga, ON, Canada) and 4l (3.125ng/l)  of invertebrate DNA is followed by amplification at 98C for 30 secs, 35 cycles of (98 C for 30 secs, 59 C for 30 secs, 72C for 30 secs) and a final extension at 72 C for 2 min. Amplicons were purified using SpriSelect (Beckman Coulter, CA, USA) with a sample to volume ratio of 0.6x and quantified. Purified amplicons were self-ligated to form plasmid like structures using Blunt/TA Ligase Master Mix (NEB, Whitby, ON, Canada) and treated with Plasmid-SafeTM ATP-dependent DNAse kit (Lucigen Corp, Middleton, WI, USA) for the removal of linear molecules. Final products were purified with SpriSelect magnetic beads at a 0.6x ratio and quantified. Rolling Circle Amplification (RCA) was performed with TruePrimeTM RCA kit following the manufacturers instructions for six aliquots of 2.5 L circularized DNA and negative controls were included using nuclease-free water instead of a cleaned product (Expedeon Corp, San Diego, CA, USA). RCA products were incubated for 2.5 to 6 hours at 30C.