# A Novel Singleton Giant Phage Yong-XC31 Lytic to the _Pyropia_ Pathogen _Vibrio mediterranean_
## 2 Materials and Methods
### Genome Annotation and Taxonomic Analyses  
ORFs prediction was initially conducted with RAST ([[http://www.rast.nmpdf.org](http://www.rast.nmpdf.org)]([http://www.rast.nmpdf.org](http://www.rast.nmpdf.org)), accessed on 30 December 2020), and then identified with HMMER and HHpred web server [29; 30]. All predicted ORFs were manually verified using the BLAST tool of NCBI ([[https://blast.ncbi.nlm.nih.gov/](https://blast.ncbi.nlm.nih.gov/)]([https://blast.ncbi.nlm.nih.gov/](https://blast.ncbi.nlm.nih.gov/)), accessed on 30 December 2020). tRNAscan-SE was used to search for tRNA genes ([[http://lowelab.ucsc.edu/tRNAscan-SE/](http://lowelab.ucsc.edu/tRNAscan-SE/)]([http://lowelab.ucsc.edu/tRNAscan-SE/](http://lowelab.ucsc.edu/tRNAscan-SE/)), accessed on 30 December 2020; [31]), and the antibiotic resistance genes and virulence factors were searched in CARD \begin{table}
\begin{tabular}{c c c c} \hline \hline
**No. Bacteria Strains** & **Lytic Ability** & **No. Bacteria Strains** & **Lytic Ability** \\ \hline _Vibrio mediaterrane_ 117-T6 & + & _Escherichia coli_ & DH5\(\alpha\) & - \\ _Vibrio hisiensis_ XXV & - & _Citrobacter freundini_ & - \\ _Vibrio bratisalis_ XOY1 & - & _Shevnella putredaciens_ & - \\ _Vibrio unlificus_ XXY & - & _Strupococcus innate_ & - \\ _Vibrio bratisalis_ XOY2 & - & _Alternate pseudomonas_ XY & - \\ _Vibrio parahanehydricus_ IA08161 & - & _Sibaglia dysenteric_ & - \\ _Vibrio urananehydricus_ IA11665 & - & _Protein vulgaris_ & - \\ _Vibrio alginolyticus_ LDF & - & _Proteins mintilis_ & - \\ _Vibrio alginolyticus_ XY & - & _Aeromonas hydrophilla_ & - \\ _Vibrio alginolyticus_ WY & - & _Sibaglia somer_ & - \\ _Vibrio alginolyticus_ SZT & - & _Pseudomonas aeruginosa_ LDF & - \\ _Vibrio harvegi_ 1-5 & - & _Pseudomonas aeruginosa_ SZT & - \\ _Vibrio harvegi_ LDF & - & _Aeromonas sodruix_ IX-23 & - \\ _Vibrio paucini_ XY & - & _Aeromonas sodruix_ ATC43979 & - \\ _Vibrio angullatum_ & - & _Pseudallermous isoschenchitti_ XY & - \\ _Aeromonas bivuluum_ XXY & - & _Salmonella parahpuli_ B & - \\ _Edwardsiella tarda SZT_ & - & _Entreobacter claacea_ & - \\ _Edwardsiella tarda_ LW & - & _Martinomonas_ sp.XY & - \\ _Entreobacter sakaziki_ & - & _Pseudomonas_ sp.XXY & - \\ \hline \hline \end{tabular} (+) representative infection, (-) representative non-infection.  
\end{table} 
Table 1: Bacterial strains used in the host range assay and infection results. database ([[http://arpcard.mcmaster.ca](http://arpcard.mcmaster.ca)]([http://arpcard.mcmaster.ca](http://arpcard.mcmaster.ca)), accessed on 30 December 2020) and VFDB database ([[http://www.mgc.ac.Cn/VFs/main.htm](http://www.mgc.ac.Cn/VFs/main.htm)]([http://www.mgc.ac.Cn/VFs/main.htm](http://www.mgc.ac.Cn/VFs/main.htm)), accessed on 30 December 2020), respectively. Nucleotide sequence comparisons were firstly conducted using NCBI BLASTn[32]. The pair-wise average nucleotide identity (ANI) values between the giant phage Yong-XC31 and the phages with the 10 top highest homology (Table 2) in BLASTn comparison (e-value < 10\({}^{-5}\)) were confirmed using OrthoANI [33]. The in silico DNA-DNA hybridization (isDDH) values between Yong-XC31 and these closely related giant phages were performed using GGDC (formula 2) [34]. The percentage of conserved proteins (POCP) values between Yong-XC31 and these related giant phages were calculated as described previously [35]. Genome comparison of Yong-XC31 and the closest related phage BONAISHI was done by a genome comparison visualizer, Easyfig [36]. To estimate the nucleotide sequence similarity between Yong-XC31 and other phages in current (26 January 2021) public databases, the Pairwise Sequence Comparison (PASC) classification tool [37] ([[https://www.ncbi.nlm.nih.gov/sutils/pasc/virridty.cgi](https://www.ncbi.nlm.nih.gov/sutils/pasc/virridty.cgi)]([https://www.ncbi.nlm.nih.gov/sutils/pasc/virridty.cgi](https://www.ncbi.nlm.nih.gov/sutils/pasc/virridty.cgi)), accessed on 30 December 2020) was used.