Illumina Seq of water eDNA: AM1
Samples were prepared for Illumina Sequencing by COI gene amplification of the eukaryotic communities. The DNA was amplified for the hypervariable region with specific primers and further reamplified in a limited-cycle PCR reaction to add sequencing adapters and dual indexes. First PCR reactions were performed for each sample using KAPA HiFi HotStart PCR Kit (Kapabiosystems, Cape Town, South Africa) according to manufacturer suggestions. Second PCR reactions added indexes and sequencing adapters to both ends of the amplified target region according to manufacturers recommendations (Illumina, 2013). PCR products were then one-step purified and normalized using SequalPrep 96-well plate kit (ThermoFisher Scientific, Waltham, USA), pooled and pair-end sequenced in the Illumina MiSeq sequencer with the MiSeq reagent Kit v3 (600 cycles), according to manufacturers instructions (Illumina, San Diego, CA, USA) at Genoinseq (Cantanhede, Portugal).