eDNA metabarcoding of UK lake communities: Windermere_33-CytB
Samples were PCR amplified with a one-step library preparation protocol using individually tagged forward and reverse primers allowing for 96 uniquely dual-indexed combinations (Kozich et al. 2013). PCRs were replicated three times for each sample and pooled in order to minimise bias in individual PCR reactions. PCRs were performed in 25 _l volumes with Q5 High-Fidelity PCR Kit (New England Biolabs, UK) containing: 1X Master Mix, 0.5 _M of each tagged primer and 2.5 _l template DNA. PCRs were performed on an Applied Biosystems Veriti Thermal Cycler with the following profile: 98 ¡C for 30 min, 40 cycles of 98 ¡C for 10 sec, 50 ¡C for 15 sec and 72 ¡C for 20 sec, followed by a final elongation step at 72 ¡C for 5 min. 12S primers: 12S_F1 - 5'-ACTGGGATTAGATACCCC-3'; 12S_R1 - 5'-TAGAACAGGCTCCTCTAG-3' (Kelly et al. 2014). CytB primers: CytB_L14841 - 5'-AAAAACCACCGTTGTTATTCAACTA-3'; CytB_15149R - 5'-GCDCCTCARAATGAYATTTGTCCTCA-3' (Kocher et al. 1989). Each library was normalised and samples subsequently pooled. Libraries were run at a 6 pM concentration on an Illumina MiSeq using the 2 x 300 bp V3 chemistry. In order improve clustering during the initial sequencing cycles 10% of PhiX genomic library was added.