Mesophotic environmental DNA: fishes of BT201_12
Library preparation was performed as a two-step PCR using AccuPower ProFi Taq PCR PreMix (Bioneer: California, USA) and the MiFish-U teleost-specific primers (12S ribosomal RNA gene 163-185 bp; Miya et al., 2015). The first step PCR was performed using 1 l of 10 M of both the forward and reverse primers combined with sample volume that equated to 500 ng of DNA. Each reaction was filled up to 20 l. A SimpliAmp Thermal Cycler (Applied biosystems: Foster City, California, USA) was used for the PCR, with the following thermal profile an initial denaturation at 94C for 3 minutes followed by 30 cycles of: 30 seconds at 94C; annealing at 60C for 30 seconds; extension at 69C for 30 seconds and a final extension period at 69C for 7 minutes. For confirmation of successful PCR, the product was run on 2 % agarose gel with a Quick-Load Purple Low Molecular Weight DNA Ladder (New England Biolabs: Massachusetts, USA). PCR product was diluted (1:10) with DNA free water and 10l used as the template for the second PCR step, combined with 1L of 10 M forward and reverse dual Illumina index primers in a total reaction volume of 20 l. The reaction thermal profile followed step one. Each second step PCR was completed in triplicate with reactions pooled to give a final volume ~55 l (after 5 l were used for gel electrophoresis). Sequencing protocols were as follows: library concentration was determined using Qubit Fluorometer (Thermo Fisher Scientific) and quality assessed with an Agilent Fragment Analyser (Agilent, Santa Clara, CA). The library was normalized to equimolar concentrations alongside another one from a separate, concomitant study, and both libraries were then pooled for size selection (200  400 bp) using a Pippin HT (Sage Science). To normalise pooled libraries, concentrations were confirmed, and quality assessed using a Qubit Fluorometer (Thermo Fisher Scientific) and an Agilent Fragment analyser (Agilent, Santa Clara, CA). Sequencing was performed using a V2 Reagent Kit and standard flow cell with paired end reads of 250 bp with custom sequencing primers on an Illumina MiSeq platform.