Method
1 Genome
A total of 32 genomic sequences of ranaviruses from various cold-blooded
vertebrates (reptiles, amphibians and fishes) were obtained from NCBI
(National Center of Biotechnology Information). The detailed information
about host species, the country of origin and the year of detection are
listed in Table S1.
2 Core-pan analysis
In order to identify the core genes of ranavirus, the pan-genome
analysis of orthologous gene clusters were carried out using PanX(Ding
et al., 2017) for the 32 completed ranavirus genomes. The strictly core
genes (including genes present in all viral genomes) were identified
using PanX(Ding et al., 2017) with the parameters as “-dmi 0.7” and
“-nsl”. “-dmi 0.7” means that the sequence identity threshold to
report an alignment is 0.7, and “-nsl” means to “disable long branch
splitting”.
3 Phylogenetic analysis of
genome
Composition vector phylogenetic tree (CV-Tree)(Gao & Qi, 2007; Zuo &
Hao, 2015): the amino acid sequences in FASTA format were directly
summited in CVTree3 Web Server
(http://tlife.fudan.edu.cn/cvtree/cvtree/) and K-tuple length was set at
6. Additionally, the genomic sequences of other Iridovirus family were
included as an out-group. Visualization and annotation for phylogenetic
tree were performed using Evolview v3 (Subramanian et al., 2019).
Neighbor-joining phylogenetic tree (NJ-Tree) based on ranavirus core
genes: the ranavirus core genes identified by core-pan analysis were
aligned using MAFFT software (Katoh et al., 2009), and the aligned
sequences were concatenated in order using PhyloSuite (Zhang et al.,
2018). Finally, the streamlined sequences were used to construct
Neighbor-joining phylogenetic tree by using MEGA 6.0 with 1,000
bootstrap replicates(Tamura et al., 2013). NJ-Tree based on iridovirid
core genes: iridovirid core genes were selected from the ranavirus core
genes identified by core-pan analysis. The subsequent steps were same as
Neighbor-joining phylogenetic tree (NJ-Tree) based on ranavirus core
genes.
4 Dot plot analysis
Dot plot analysis of ranaviruses was performed using Java Dot Plot
Alignments (JDotter) (Brodie et al., 2004). FV3 (NCBI access number is
AY548484) was placed in horizontal sequence, meanwhile the other
ranaviruses were placed in vertical sequence. Maximum plot size was set
at 700 bases and sliding window size was set at 50. The nucleic acid
sequences of FASTA format used for Dot plot analysis were obtained from
NCBI.
5 Recombination analysis
The ranavirus core genes identified by core-pan analysis were
concatenated using PhyloSuite (Zhang et al., 2018). The streamlined
sequences were imported into the Recombination Detection Program (RDP)
BETA4.67 (Darren P Martin et al., 2015). Putative recombination
breakpoints were visualized in RDP 4 software by algorithms of RDP,
GENECONV, Bootscan, Maxchi, Chimaera, SiSscan, PhylPro, LARD and 3Seq.
UPGMA (unweighted pair group method using arithmetic average)
phylogenetic tree was constructed by TREES module within RDP4.
6 Substitution saturation
analysis
The ranavirus core genes were imported separately to DAMBE Version
5.3.19 (Xuhua Xia, 2013). The index of nucleotide substitution
saturation was estimated using Xia method (Xuhua Xia et al., 2003).