Cameroonian fruit bats harbor divergent viruses, including rotavirus H, bastroviruses, and picobirnaviruses using an alternative genetic code

Yinda, Claude Kwe; Ghogomu, Stephen Mbigha; Conceição-Neto, Nádia; Beller, Leen; Deboutte, Ward; Vanhulle, Emiel; Maes, Piet; Van Ranst, Marc; Matthijnssens, Jelle

NGS reads were analyzed as described in (Yinda et al. 2016a,b) Briefly, after raw reads were trimmed and de novo assembled using trimmomatic and SPAdes, respectively (Bankevich et al. 2012; Bolger et al. 2014) , the assembled contigs were annotated by DIAMOND with the sensitive option using the GenBank's non-redundant (nr) database (Buchfink et al. 2015) . From the contigs, ORFs were identified and further analyzed for conserved motifs identification in the amino acid sequences using NCBI's conserved domain database (CDD) (Marchler-Bauer et al. 2015) and/or Pfam (Finn et al. 2014 ). To rule out the possibility of false positive, the presence of some of the viruses were confirmed on original samples by PCR (bastrovirus, CoV (66/2014/ CMR, N704-P13), densovirus (CAMBtDV2, CAMBtDV4) and RVH; list of primers in Supplementary Table S2 ). Additionally, we checked for common contigs across our NGS runs which may indicate contamination from other samples or commercial kits and these were excluded from the analysis. Amino acid alignments were used for all trees except CoV and RVH alignments and for each of these nt alignments, a test for substitution saturation was performed using Dambe (Xia 2017) . Alignments of viral sequences were made with Muscle implemented in MEGA7 (Molecular Evolutionary Genetics Analysis version 7) (Kumar et al. 2016) or MAFFT (Multiple Alignment using Fast Fourier Transform) (for Picobirnaviridae and Partiviridae, because of the high genetic diversity in these families) (Katoh et al. 2002) . After trimming with trimAL (Capella-Gutié rrez et al. 2009), substitution models were determined using ModelGenerator (Keane et al. 2006 ) and phylogenetic trees constructed using RAxML (Stamatakis 2014) , with the autoMRE flag, which enables a posteriori bootstopping analysis. All trees were visualized in FigTree (http://tree.bio.ed.ac.uk/software/figtree/) and midpoint rooted for purposes of clarity. Only bootstrap values >70% are shown except at branches and clusters including the described bat virus. The choice of the sequence type (nucleotide or amino acid) for alignment and phylogenetic analysis was based on the genetic relatedness between the novel strains and reference strains, and the classification criteria of the viral family/genera to which the virus strain belongs.