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In the second PCR, 3 L of the first PCR reaction product was used as the template and was combined with 2U of Taq, 1 buffer, 1.5 mM MgCl 2 , 0.4 mg/mL BSA, 0.2 mM dNTPs, and 5pmols each of Illumina adapted primers 341F (CCTACGGGAGGCAGCAG) and 518R (ATTACCGCGGCTGCTGG) (primers + Illumina adapters/barcode/priming region as described in supplemental materials of Bartram et al., 2011: ~ 80 bp) 44,45,46