Next generation sequencing of arthropod prey from primate fecal samples
I used full plate PCR to amplify a ~157 bp region of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene using modified arthropod-specific primers (ZBJ-ArtF1c: AGATATTGGAACWTTATATTTTATTTTTGG, ZBJ-ArtR2c: WACTAATCAATTWCCAAATCCTCC) (Madden et al. 2016). Primers were modified to include adapters for multiplex sequencing on the Illumina MiSeq platform, and the reverse primer for each sample was tagged with an individual error-correcting 12-basepair barcode. PCR amplification was performed in a total volume of 25 ?L, including 3 ?L gDNA, 12.5 ?L Master Mix (Promega Biotech Co.), and 1 ?M each of the forward and reverse primers. Cycling conditions followed Madden et al. (2016) (94 C for five min, followed by 45 cycles of 94 C 30 s, 45 C 45 s, 72 C 45 s and a final extension at 72 C for 10 min). PCR amplicons were visualized on 2% agarose gels, and 2-3 replicate PCR reactions were conducted per extracted DNA sample. Barcoded PCR products for each sample were pooled, cleaned, and normalized with SequalPrep Normalization Plates (Thermo Fisher Scientific). PCR products were then pooled across samples and sequenced on a single Illumina MiSeq run using 2x150bp paired-end chemistry
SUB5156450
ARTHRO