Mecagenomics of fecal samples of Lycalopex gymnocercus, cold season
Metagenomic barcoding of mitochondrial 12S rRNA with blocking oligonucleotides. The fox- blocking oligonucleotide developed with a phosphate (PHO) 3' extreme modification (5-CCACTATGCTTAGCCTTAAACGTAAATGATTYTAT PHO- 3') was included in the 12S fragment PCR from fecal DNA (Section 2.2), using the primer set 12S-V5 (29). This 12S rRNA gene fragment was used in carnivores and omnivorous DNA metabarcoding feeding studies previously (2,10,28,39).The PCR consisted ofin 10l final volume with 0.04U of Taq polymerase (Invitrogen, USA), 1X Taq buffer, 1.5mM of MgCl, 0.2mg/mL of BSA (Bovine Serum Albumin), 0.05mM of dNTPs, 0.1 M of forward and reverse primers, 1.6M of fox blocking primer and a 4-8ng/l final concentration of DNA. The primers 12S-V5 forward and reverse for the 12S fragment mtDNA gene contained overhang sequences that were used on Aa second round of indexing PCR amplifications was done to add the Nextera indexes for sequencing on a MiSeq Illumina platform. The thermal profile started with a denaturalization step of 94C for 3 minutes, followed by 35 cycles of 94C for 30 seconds and 58C for 90 seconds. We used 40 to 45 cycles in degraded fecal DNA. Positive and negative controls were included inon each PCR reaction. Amplification success was confirmed by visualizing PCR products in 2X agarose electrophoresis using Goodview (SBS Genetech, Beijing) nucleic acid stain. Each PCR productsproducst were then obtained was purified using 0.8U/l of Exonuclease I and 0.2U/l of Thermosensitive Alkaline Phosphatase (FastAP) enzymes (ThermoFisher Scientific, USA); the thermal profile was 37C for 1 hour and 30 minutes followed by 5 minutes at 75C. PCR-purifiedPCR purified products were quantified using a Nanodrop TMND-1000 UV-vis Spectrophotometer (Nano-Drop Technologies, Inc., Wilmington, DE, USA) and then. PCR purified products from the same fox species and season were pooled in equimolar concentrations. Thus, four pools were generated that included libraries prepared from crab-eating and pampas foxes'foxes feces collected in warm and cold seasons. Paired-end (2X 100) sequencing was performed using the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA), available at the sequencing facility of the Institut Pasteur, Montevideo.