carrion fly gut contents for vertebrate metabarcoding
PCR was performed on all samples using a mammal specific primer set targeting 130-138bp of the mitochondrial 16S rRNA locus (Boessenkool et al. 2012), and a pan-vertebrate primer set targeting 140-143bp of the mitochondrial 12S locus. Reactions also included human blocking primers (Boessenkool et al. 2012) and Sus blocking primers to decrease competition from contaminating DNA from the pork bait (Calvignac-Spencer et al. 2013b). Both 16S and 12S primers included a 5 addition of a 33bp Illumina specific sequence for addition of illumina adapters. A minimum of two PCR replicates per sample were performed in 10 ?l volumes that for 16S included 0.2 um of each primer, 1 ?m of human blocking primer (5x) 4 ?m of Sus blocking primer (20X), 200 ?m dNTP,  4 mM MgCl2, 1X PCR buffer,  1.25 U PlatinumŽ Taq polymerase (Invitrogen) and 3 ?l of template DNA. For 12S, volumes were the same, except MgCl2 was reduced to 2.5 mM. Cycling conditions were 10 min at 95 °C, followed by 42 cycles (16S) or 47 cycles (12S) of 30 s at 95 °C, 30 s at 64 °C, and 1 min at 72 °C, with a final extension of 10 min at 72 °C. Following the initial PCR, sample replicates were pooled, and a second PCR was conducted to add Illumina flow cell binding sequences, and unique, 8bp sample specific indexes to both ends of each amplicon. Reaction included 1 ?l each of a unique forward and unique reverse illumina index tag, 2.5ul of 10X Qtaq buffer, 1.5Mm MgCl2, and 1.25 units of Qiagen Qtaq. Cycling conditions were 3 min at 94°C, followed by 6 cycles of 45 s at 94 °C, 60 s at 50 °C, and 1 min at 72 °C, with a final extension of 10 min at 72 °C. All PCRs included no-template negative controls to monitor for contamination. Samples were purified and normalized using SequalPrep Normalization Plates (Thermo-Fisher Scientific; Waltham, MA). All samples were pooled in equimolar concentration using 5 ?l of each sample and pools were then concentrated using standard Ampure XP beads (Beckman Coulter; Indianapolis, IN). The concentrated pool was quantified using a Qubit fluorometer and quality checked using a Bioanalyzer High Sensitivity Kit. The library was then sequenced on an Illumina MiSeq using a 2 x 250 PE sequencing kit.