Non-invasive surveys of mammalian viruses using environmental DNA

Niccolo Alfano; Anisha Dayaram; Jan Axtner; Kyriakos Tsangaras; Marie-Louise Kampmann; Azlan Mohamed; Seth Timothy Wong; M. Thomas P. Gilbert; Andreas Wilting; Alex Daivd Greenwood

All Coronaviridae contigs matched a bat betacoronavirus as determined by BLAST searches with identities between 70-73% (Suppl. Tab. 1). The resulting sequence did not cluster in any of the four clades representing the known Coronaviridae genera, suggesting it may represent a novel coronavirus genus (Suppl. Fig. 4 ). Each contig overlapped with the coronavirus RNA-dependent RNA polymerase gene (orf1ab), the viral region mainly targeted by the RNA oligonucleotide baits (Suppl. Anelloviridae contigs matched either porcine torque teno virus (PTTV) (95-96% identity), a giant panda anellovirus (GpAV) (81-92% identity) or a masked palm civet torque teno virus (Pl-TTV) (83-92% identity) (Suppl. Tab. 1). The PTTV contigs were found in two samples (L8 and L37), while the GpAV and Pl-TTV contigs were detected in six samples. GpAV was the best match in four samples (L7, L17, L36, L39) and Pl-TTV in three (L21, L25, L39). In sample L39 both were identified. Every Anelloviridae contig mapped to the non-coding region of the relative reference genome since all Anelloviridae baits targeted the same untranslated region (Suppl. Fig. 6A , C, E). The non-coding region sequenced is not phylogenetically informative and therefore, phylogenetic analysis could not be performed. Viral contigs were confirmed by PCR and Sanger sequencing for samples L7, L17, L25 and L37 (Suppl. Fig. 6B , D, F).