Results of a mammal metabarcoding analyses of duiker feces collected in Taï National Park, Côte d'Ivoire

To explore how duikers might be exposed to MPXV, we examined their diet in Taï National Park, Côte d’Ivoire, with the aim of identifying trophic links that could allow for transmission. Duikers are generally considered mostly herbivorous, but sporadic observations and camera trap footage of scavenging on mammal carcasses suggest a carnivorous component to their diet. We applied a mammal-generic metabarcoding assay to duiker pellets collected between July 2017 and February 2020 in TNP (N=146), finding that more than 21% contained non-duiker mammal DNA (N=31). To detect non-host mammal DNA, we applied a three-step 16S metabarcoding protocol designed to reduce amplification bias. A 130 bp mitochondrial 16S fragment was amplified using mammal-generic primers 16S mam1/16S mam2 together with blocking primers against human, pig, and host (cattle) DNA. PCRs were run in quadruplicates with extraction blanks and no-template controls processed throughout. Amplicons of the expected size were gel-purified (Qiagen QIAquick), bead-cleaned (AMPure XP, 1.8×), dual-indexed (Nextera XT), and sequenced on an Illumina NextSeq 500 (2 × 150 bp). Negative controls were sequenced alongside samples to monitor potential cross-contamination. For the duiker samples which were processed with untagged primers, we considered a detection event as genuine if it appeared in at least 10 reads and made up a minimum 1% of the total assignable reads. One contaminated extraction control contained mammal DNA, so all co-processed samples were excluded (N=15); thus, we were able to examine the presence of non-duiker DNA in 146 duiker samples. In addition, those taxa found in the contaminated control (Cercopithecus diana, Piliocolobus badius and Procolobus verus) were not considered as detection events in any of the other samples.