Dietary DNA metabarcoding of small-herbivore fecal samples
We amplified the P6 loop of the chloroplast trnL (UAA) intron. We performed PCR in 25 ?L reactions using 1.5 mM MgCl2, 200 ?M each dNTP, 0.2 ?M each primer [trnL(UAA)c/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 30 s, and 72C for 1 min, with a 10 min final extension at 72C. We verified PCR success by running 3 ?l of PCR product on a 1.5% 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 250 x 250 bp paired-end chemistry.