DNA metabarcoding of large-herbivore fecal samples
We amplified the P6 loop of the chloroplast trnL (UAA) intron. We performed PCR in 25 L reactions using 2 mM MgCl2, 200 M each dNTP, 0.2 M each primer [trnL(UAA)g/trnL(UAA)h], Platinum Taq DNA polymerase, and 2 L of DNA extract. The g and h primers were modified to contain an adapter overhang. Thermocycling followed a program of initial denaturation at 95C for 5 min, followed by 35 cycles of 95C for 30 s, 55C for 40 s, and 72C for 1 min, with a 10 min final extension at 72C. We verified PCR success by running 5 l of PCR product on a 2.0% agarose gel. Initial amplicons were then sent to the University of Rhode Island sequencing core where multiplex identification (MID) tags and P5/P7 adapters were annealed to the overhang adapter in a second-round PCR to enable Nextera Illumina sequencing. Libraries were pooled at equimolar concentrations and sequenced on the Illumina MiSeq platform with 150 x 150 bp paired-end chemistry.