Infrastructure for a Phage Reference Database: Identification of Large-Scale Biases in The

Home , Enterobacter

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 INfrastructure for a PHAge REference Database: Identification of large-scale biases in the

2 current collection of phage genomes

4 Ryan Cook1, Nathan Brown2, Tamsin Redgwell3, Branko Rihtman4, Megan Barnes2, Martha

5 Clokie2, Dov J. Stekel5, Jon Hobman5, Michael A. Jones1, Andrew Millard2*

7 1 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington

8 Campus, College Road, Loughborough, Leicestershire, LE12 5RD, UK

9 2 Dept Genetics and Genome Biology, University of Leicester, University Road, Leicester,

10 Leicestershire, LE1 7RH, UK

11 3 COPSAC, Copenhagen Prospective Studies on Asthma in Childhood, Herlev and Gentofte

12 Hospital, University of Copenhagen, Copenhagen, Denmark

13 4 University of Warwick, School of Life Sciences, Coventry, UK

14 5 School of Biosciences, University of Nottingham, Sutton Bonington Campus, College Road,

15 Loughborough, Leicestershire, LE12 5RD,

17 Corresponding author: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

18 Abstract

19 Background

20 With advances in sequencing technology and decreasing costs, the number of

21 bacteriophage genomes that have been sequenced has increased markedly in the last

22 decade.

23 Materials and Methods

24 We developed an automated retrieval and analysis system for bacteriophage genomes,

25 INPHARED (https://github.com/RyanCook94/inphared), that provides data in a consistent

26 format.

27 Results

28 As of January 2021, 14,244 complete phage genomes have been sequenced. The data set is

29 dominated by phages that infect a small number of bacterial genera, with 75% of phages

30 isolated only on 30 bacterial genera. There is further bias with significantly more lytic phage

31 genomes than temperate within the database, resulting in ~54% of temperate phage

32 genomes originating from just three host genera. Within phage genomes, putative antibiotic

33 resistance genes were found in higher frequencies in temperate phages than lytic phages.

34 Conclusion

35 We provide a mechanism to reproducibly extract complete phage genomes and highlight

36 some of the biases within this data, that underpins our current understanding of phage

37 genomes.

39 Keywords: phage genomes, antibiotic resistance genes, virulence genes, jumbo-phages bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

40 Introduction

41 Bacteriophages (hereafter phages), are viruses that specifically infect bacteria and are

42 thought to be the most abundant biological entities in the biosphere (1). In the oceans they

43 are important in diverting the flow of carbon into dissolved and particulate organic matter

44 via the lysis of their hosts (1), or directly halting the fixation of CO2 carried out by their

45 cyanobacterial hosts (2). In the human microbiome, it is becoming increasingly clear that

46 phages play a role in a range of different diseases. Many recent studies have shown disease-

47 specific alterations to the gut virome community in both gastrointestinal and systemic

48 conditions, including irritable bowel disease (3), AIDs (4), malnutrition (5), and diabetes (6).

50 Phages alter the physiology of their hosts such that their bacterial hosts display increased

51 virulence, a notable example being phage CTX into the genome of Vibrio cholerae, resulting

52 in cholera (7). However, there are many cases where the expression of phage-encoded

53 toxins cause an otherwise harmless commensal bacterium to convert into a pathogen,

54 including multi-drug resistant ST11 strains of Pseudomonas aeruginosa (8, 9), and the Shiga-

55 toxin encoding Escherichia coli (10). As well as increasing the virulence of the host bacteria,

56 phages can also utilise parts of their genomes known as auxiliary metabolic genes (AMGs),

57 homologues of host metabolic genes, to modulate their hosts metabolism (11).

59 Our understanding of how phages alter host metabolism has increased in conjunction with

60 the number of phage genomes that have been sequenced, following sequencing of the first

61 phage genome in 1977 (12). Since then, the number of phages that are isolated and the

62 relative ease of high-throughput sequencing has led to a rapid increase in the number of

63 sequenced bacteriophage genomes (13). The relatively simple nature of phage genomes bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

64 means that the vast majority of isolated phage genomes can be completely assembled using

65 short-read next generation sequencing (14). The greater number of phage genomes

66 available results in common analyses, including comparative genomic analyses (15, 16),

67 taxonomic classification of phages (17–20), forming the basis of software to predict new

68 phages (21–26), and as is often the first step in analysis of viromes, the comparison of

69 sequences to a known database.

71 To do all of the above requires a comprehensive set of complete phage genomes from

72 cultured isolates that can be used to build databases for further analyses. It also raises the

73 question of how many complete phage genomes are currently available. While this should

74 be relatively trivial question to answer, it is not very simple to do so, as there is currently no

75 such database of all complete phage genomes. Therefore, the aim of this work was to

76 provide a reproducible and automated way to extract complete phage genomes from

77 GenBank and identify general properties within the data and limitations.

79 Materials and Methods

80 Bacteriophage genomes were download using the “PHG” identifier along with minimum and

81 maximum length cut-offs. Genomes were then filtered based on several parameters to

82 identify complete and near complete phage genomes. This includes initial searching for the

83 term “Complete” & “Genome” in the phage description, followed by “Complete” &

84 (“Genome” or “Sequence”) & a genome length of greater than 10 kb. The list of genomes

85 was then manually curated to identify obviously incomplete phage genomes, with the

86 process on going. The accessions of these are then excluded in future iterations by the use

87 of an exclusion list, which can be added to by the community via GitHub. Whilst this process bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

88 is not perfect, we thank numerous people that have identified genomes within this list that

89 are obviously incomplete. The initial search term for downloading genomes was: esearch -

90 db nucleotide -query "gbdiv_PHG[prop]" | efilter -query "1417:800000 [SLEN] " | efetch -

91 format gb > $phage_db.gb. An exclusion list of phage genomes that are automatically called

92 “complete”, yet when manually checked are not is continually being updated.

94 After filtering, genes are called using Prokka with the –noanno flag, with a small number of

95 phages using –gcode 15 (27, 28). Gene calling was repeated to provide consistency across all

96 genomes, which is essential for comparative genomics. A database is provided so that this

97 process does not continually have to be rerun and only new genomes are added. The

98 original GenBank files are used to gather useful metadata including taxa and bacterial host,

99 and the Prokka output files are used to gather data relating to genomic features. The

100 gathered data are summarised in a tab-delimited file that includes the following: accession

101 number, description of the phage genome, GenBank classification, genome length (bp),

102 molGC (%), modification date, number of CDS, proportion of CDS on positive sense strand

103 (%), proportion of CDS on negative sense strand (%), coding capacity (%), number of tRNAs,

104 bacterial host, viral genus, viral sub-family, viral family, and the lowest viral taxa available

105 (from genus, sub-family and family). Coding capacity was calculated by comparing the

106 genome length to the sum length of all coding features within the Prokka output, and tRNAs

107 were identified by the use of tRNA tag. Other outputs include a fasta file of all phage

108 genomes, a MASH index for rapid comparison of new sequences, vConTACT2 input files, and

109 various annotation files for IToL and vConTACT2. The vConTACT2 input files produced from

110 the script were processed using vConTACT2 v0.9.13 with --rel-mode Diamond --db 'None' -- bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

111 pcs-mode MCL --vcs-mode ClusterONE --min-size 1 and the resultant network was visualised

112 using Cytoscape v3.8.0 (29, 30).

113

114 To identify genes indicative of a temperate lifestyle within genomes, we used a set of PFAM

115 HMMs as described previously (31). If a genome encoded one of these genes, it was

116 assumed to be temperate. Antimicrobial resistance genes (ARGs) and virulence factors were

117 identified using Abricate with the resfinder and VFDB databases using 95% identity and 75%

118 coverage cut-offs (32–34).

119

120 The phylogeny of “jumbo-phages” was constructed from the amino acid sequence of the

121 TerL protein, extracted from 313/314 of the “jumbo-phage” genomes. Sequences were

122 queried against a database of proteins from non-“jumbo-phages” using Blastp and the top

123 five hits were extracted (35) with redundant sequences being removed. Sequences were

124 aligned with MAFFT, with a phylogenetic tree being produced using IQ-Tree with “-m WAG -

125 bb 1000” which was visualised using IToL (36–38). Additional information was overlaid using

126 IToL templates that are generated via INPHARED.

127

128 Rarefaction analysis was carried out for phage genomes from the top ten most common

129 hosts (70% ID over 95% length) and species (95% ID over 95% length) using ClusterGenomes

130 v5.1 (39). An additional set of these genomes pooled together was included. Rarefaction

131 curves and species richness estimates were produced using Vegan in R (40, 41).

132 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

133 All data from January 2021 is available at Figshare

134 https://doi.org/10.25392/leicester.data.14242085 and the script used for downloading and

135 analysing genomes is available on GitHub https://github.com/RyanCook94/ . bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

136 Results

137 The output of the INPHARED script provides as set of complete phage genomes, whereby

138 genes have been called in a consistent manner that allows comparative genomics and

139 phylogenetic analysis. In addition, it provides a MASH database to allow rapid comparison of

140 new phage genomes against to identify close relatives. Along with formatted databases for

141 input into vConTACT2 to allow identification of more distant relatives. The host data (Genus)

142 for each phage is extracted along with summary information for each genome, which is

143 reformatted to allow overlay onto trees in IToL (See Supplementary Figure 1 for full details).

144

145 For this study, we used a lenient definition of “complete” for the identification of complete

146 phage genomes. Strictly speaking a complete phage genome would include the terminal

147 ends of the phage genome. As many phages are sequenced using a transposon based library

148 preparation (16, 42), the genome can never be complete as the terminal bases can never be

149 sequenced, unless it is circularly permuted (14). For phage genomes with long terminal

150 repeats, if the length of the repeat is larger than the library insert size, these cannot be

151 resolved. As this information is not included in every GenBank file, automated retrieval is

152 not possible.

153

154 We next set about identifying how many phage genomes have been sequenced to date. The

155 extraction of genomes from the nucleotide database of GenBank results in 18,134 genomes.

156 Of these, 3,890 phage genomes are REFSEQ entries which are derived from primary

157 submissions, resulting in 14,244 putative complete phage genomes. Current

158 recommendations are that phages are uniquely named (43), if this assumption is true then

159 number of unique phage genomes is 12,127 if phages with the same name are truly bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

160 identical. However, there are multiple examples of phages with the same name. In some

161 cases this is the same phage being re-sequenced due to experimental evolution studies such

162 as E. coli phiX (44) . In other instances, phages with the same name are not genetically

163 identical. Thus, using a phage name as a means to identify different phages is not a suitable

164 method for determining the number of unique phage genomes. As an alternative, de-

165 duplication of genomes at 100%, 97% and 95% identity results in 13,830, 12,845 and 12,770

166 genomes respectively.

167

168 Having established a dataset of “complete” phage genomes, we then analysed this data to

169 look at general phage genomic properties. First, we looked at the increase in the number of

170 phage genomes that are sequenced over time. Whilst the number of phage genomes has

171 rapidly increased over the last 20 years, the rate of increase has slowed in the last decade

172 (Figure 1), with the number of phage genomes doubling every 2-3 years.

173

174 Bacteriophage Hosts and Predicted Gene Function

175 Utilising the database of complete genomes, we extracted the hosts and predicted number

176 of hypothetical proteins for each phage. Across all phages, the majority of genes which

177 encode proteins with unknown function (hypothetical) was mean 56% (+/- 20), supporting

178 the truism that the majority of genes encode proteins within unknown function.

179

180 The host of 12,403 phages were extracted with the remainder unknown as the host was not

181 clear from the information contained within the GenBank file alone. The genomes of phages

182 infecting 234 hosts have been sequenced. However, there is a clear bias in the isolation of

183 phages against the same host (Figure 2a). Phages that infect Mycobacterium spp. are the bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

184 most commonly deposited genomes (~13%), largely due to the pioneering work of the SEA-

185 PHAGES program (45), followed by Escherichia spp., Streptococcus spp., and Pseudomonas

186 spp. (Figure 2a). Phages isolated on just 30 different bacterial genera accounts for ~75% of

187 all phage genomes in the database (Supplementary Table 1). For genomes isolated against

188 the top ten hosts, we used rarefaction analysis to gain an understanding of the diversity of

189 phage genomes isolated to date and determine redundancy of phages isolated on a

190 particular host. Using a cut-off of 95% identity to define a species, it was clearly observed

191 the number of phage species continues to increase with the number of genomes

192 sequenced, a pattern also observed at the level of genus (70% identity) (Figure 3). Using the

193 current data, it was possible to estimate how many different species of phage might infect

194 these different hosts (Supplementary Table 4). For Mycobacterium, for which most phages

195 have isolated on, there are 695 observed species with an estimated 2132-2282 total species.

196 Thus, demonstrating even for hosts where thousands of phages have been isolated, we are

197 only just scratching the surface of the diversity of total phage diversity. We are also likely

198 under estimating the total number of different phage species. In the case of phages

199 infecting Mycobacterium, the majority of these have been isolated on only a single strain as

200 part of the SEA-PHAGES program. Increasing the diversity of the host Mycobacterium, is

201 likely to lead to higher estimates.

202

203 Lytic and Temperate phages

204 To identify if the phage is lytic or temperate, we searched for genes that facilitate a

205 temperate lifestyle (e.g., integrase and recombinase) that have been used in previous

206 studies. This process is not perfect, as the presence of an identifiable gene linked to

207 temperate phages does not mean it will access a lysogenic cycle. However, it does allow bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

208 large scale comparative analyses, compared to the manual searching of literature of every

209 phage compared to determine if it has been experimentally tested. Within the dataset,

210 4,258 (~30%) phages have the potential to access a lysogenic lifecycle. The frequency of

211 putative temperate phages was highly variable depending on the host (Supplementary

212 Figure 2). The number of putative temperate phages is also biased towards a small number

213 of hosts with 1,217, 846 and 214 isolated on Mycobacterium, Streptococcus and Gordonia

214 respectively. Collectively these three hosts account for ~54% of all putative temperate

215 phage genomes sequenced to date (Supplementary Figure 2).

216

217 Genomic Properties

218 Phage genomes ranged from 3.1 kb to 642.4 kb in size, with a clear distribution in the size of

219 genomes with the most prominent peaks at 5-10 kb, 40 kb, 50 kb and ~165 kb (Figure 2b).

220 The mean and median coding capacity was found to be 90.45% and 91.52%, respectively

221 (Supplementary Figure 2). Of the 14,244 genomes, 5,731 (~40%) were found to have ≥ 90%

222 of coding features on one strand and 3,293 (~23%) of these had coding features entirely on

223 one strand (Supplementary Figure 2). The number of phages with genes encoding tRNAs

224 was 4,590 (~32%). For those phages encoding tRNAs, the range was 1 to 62 with a median of

225 3. Whilst there is much literature on the presence of tRNAs in phages, it is still not clear

226 entirely what role they provide to phages and why they absent in some phages and not

227 others (46).

228

229 Phages with genomes greater than 200 kb are often referred to as “jumbo-phages” and are

230 reported to be rarely isolated (47). 314 genomes (~2.2%) greater than 200 kb in length were

231 identified, suggesting that they are rare. To further investigate if “jumbo-phages” are as rare bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

232 as is thought, we looked at the distribution in the context of the previously identified host

233 bias. “Jumbo-phages” have only been isolated on 31 of 234 identifiable bacterial hosts

234 (Supplementary Table 1) and are far more commonly isolated on some hosts than others.

235 Noticeably absent are any “jumbo-phages” that infect Mycobacterium, Gordonia,

236 Lactococcus, Arthrobacter, and Streptococcus, with >4,000 phages having been sequenced

237 from these bacterial hosts (Figure 2c). For host bacteria that have had far fewer phages

238 isolated on them such as Caluobacter, Sphingomonas, Erwinia, Areomonas, Dickeya and

239 Ralstonia, the frequency of “jumbo-phage” isolation is far higher (Figure 2c). Due to the

240 small sampling depth of some of these hosts (e.g., Photobacterium and Tenacibaclum), it is

241 not possible to determine whether the high proportion of genomes is merely a result of the

242 low number of genomes sequenced. However, for other hosts such as Aeromonas, Erwinia

243 and Caulobacter from which more than 20 phages have been isolated, ~26%, ~44% and

244 ~63% are categorised as “jumbo” respectively. Therefore suggesting “jumbo-phages” are

245 not always rare on particular hosts.

246

247 We further investigated the phylogeny of “jumbo-phages” using the translated sequence of

248 the terL gene. The “jumbo-phages” are well distributed across the tree and do not form a

249 single monophyletic clade, suggesting that they have arisen on multiple occasions, with

250 multiple clades of phages having representatives of “jumbo-phages” within them. Not all

251 “jumbo-phages” are equal, with “jumbo” cyanophages infecting the cyanobacteria

252 Synechococcus and Procholorococcus only marginally larger than there non-jumbo

253 cyanophages relatives. These “jumbo-phages” are also more closely related to their non-

254 jumbo cyanophages relatives than other “jumbo-phages” (Figure 4). This is not limited to

255 the cyanophages, with many other “jumbo-phages” more closely related to a non-jumbo bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

256 phage. A similar pattern of grouping non-jumbo with “jumbo-phages” is observed when a

257 reticulate approach is used to look at the relatedness of phage genomes using vConTACT2

258 (Supplementary Figure 3).

259

260 Virulence Factors and Antimicrobial Resistance Genes

261 The presence of ARGs and virulence factors is major concern for phage therapy, as the use

262 of phages carrying such genes may make the populations of bacteria they are intended to

263 kill more virulent or resistant to antibiotics. We therefore used this database to integrate

264 the frequency and diversity of phage-encoded virulence factors and ARGs. 235 genomes

265 (~1.6%) were found to encode a virulence factor and 43 genomes (~0.3%) to encode an

266 ARG. The most common virulence genes were the stx2A (72 genomes) and stx2B (71

267 genomes) genes that encode subtypes of the Shiga toxin (Supplementary Table 2). The most

268 common ARGs were the mef(A) (14 genomes) and msr(D) genes which confer resistance to

269 macrolide antibiotics (Supplementary Table 3) (48). Most genomes encoding a virulence

270 factor were predicted to be from temperate phages (222/235), and were found to infect six

271 bacterial genera, with the three most abundant hosts being Streptococcus, Staphylococcus

272 and Escherichia respectively. The hosts for many genomes could not be determined

273 (55/235). The virulence factor encoding genomes were widely distributed over 26 putative

274 genera (Supplementary Figure 3). All genomes encoding an ARG were predicted to be

275 temperate and were found to be isolated from eight bacterial genera, with the majority of

276 phages linked to Streptococcus spp. (27/43).

277

278 Discussion bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

279 Defining how many different complete phage genomes have been sequenced is not a simple

280 question as it might appear. Based on accession numbers, there are 14,244 phage genomes,

281 once RefSeq duplicates have been removed. Using unique names results in 12,127 phages,

282 however using names alone does not give an accurate estimate of the number of different

283 phages, as genomically different phages have the same name. The use of de-duplication at

284 100% identity suggests 13,830 unique phage genomes (January 2021) from cultured

285 isolates. This assumes that the genome submissions are from isolates and not predictions of

286 prophages from bacterial genomes. For the vast majority of phages, this appears to be case,

287 although not easily discernible for all phage genomes.

288

289 The data reveals clear patterns in phage genomes and biases in the selection of phage

290 genomes that are currently available, but not always discussed in the analysis of genomes.

291 The first is the number of phage genomes is relatively small. Even for hosts where the

292 highest number of phages have been isolated on, our estimates suggest 1000s of new phage

293 species remain to isolated and sequenced. If we consider there are now more than 300,000

294 assembled representative bacterial genomes in GenBank, with many hundreds of thousands

295 more for particular genera e.g., >300,000 Salmonella and Escherichia genomes alone (49).

296 The representation of phage genomes to date is tiny compared to their bacterial hosts.

297 Furthermore, the rate at which phage genomes are being sequenced is slowing down rather

298 than increasing. Given the renewed interest in phages and increased accessibility of

299 sequencing, the decrease in the rate over time was surprising.

300

301 The second point of note is the bias in phage genomes. With a clear bias in both the hosts

302 phages are isolated on and for lytic phages over temperate phages. Thus, these phages are bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

303 representative of these particular hosts, rather than phages in their entirety. Due to the

304 enormous success of the SEA-PHAGES program, many phages have been isolated on

305 Mycobacterium and Gordonia (50). This in turn results in ~1/3rd of all temperate phage

306 genomes being isolated on these two bacterial genera, whereas the remaining 2/3rds are

307 distributed across 142 different hosts.

308

309 The overrepresentation of phages infecting particular hosts can lead to truisms that may not

310 be correct. For instance, “jumbo-phages”, those that have genomes >200 kb, are rarely

311 isolated (47). Analysis of the complete dataset suggests ~2.2% of genomes fall into this

312 category. However, this needs to be viewed in the context of the large bias in the hosts used

313 for isolation, with ~75% of phages isolated on only ~16% of bacterial hosts that could be

314 identified. When the number of “jumbo-phages” is expressed as a percentage of all phage

315 genomes, their isolation is clearly rare. For some hosts, such as Mycobacterium, many

316 hundreds of phages isolated on the same host strain have been sequenced without the

317 isolation of a “jumbo-phage”, suggesting they are truly rare for this host (45). However, for

318 other hosts such as Procholorococcus, Synechococcus, Caulobacter, and Erwinia, the

319 isolation of “jumbo-phages” is not a rare event. While methodological adjustments of

320 decreasing agar viscosity and large pore size filters may increase the number of phages

321 isolated that have larger genome sizes (47), we suggest that using a wider variety of hosts

322 may increase the number of “jumbo-phages” isolated. Phylogenetic analysis demonstrated

323 many “jumbo-phages” are more closely related to non-jumbo phages than other “jumbo-

324 phages”. Thus, as the number of phage genomes has increased an arbitrary descriptor or

325 “jumbo” for phages with genomes over 200 kb in length has less meaning. Recent

326 comparative analysis of 224 “jumbo-phages”, used proteome size and analysis of protein bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

327 length to determine a cut-off of 180 kb to separate “jumbo-phages”, from other phages.

328 From this using a clustering-based approach, three major clades of “jumbo-phages” were

329 identified (51). In this study using terL as a phylogenetic marker to determine the phylogeny

330 of 313 “jumbo-phages” and their closely related phages, suggests they have arisen on

331 multiple occasions, as has been demonstrated previously (51). “Jumbo-phages” are clearly

332 not monophyletic and what applies to one “jumbo-phage” does not hold true for many

333 others (51). As the number and diversity of “jumbo-phages” increases, the use of the term

334 seems to have less meaning.

335

336 With the increasing interest and use of phages for therapy, the isolation of phages that do

337 not contain known virulence factors or ARGs is imperative. How frequently phages encode

338 antibiotic resistance genes is a topic of much debate (52, 53). A previous study of 1,181

339 phage genomes found that they are rarely encoded by phages with only 13 candidate genes,

340 of which four where experimentally tested and found to have no functional antibiotic

341 activity (47). We estimate ~0.3% of phage genomes encode a putative ARG (none have been

342 experimentally tested), a finding that is consistent with previous reports of low-level

343 carriage in phage genomes (52) in a dataset that is ~10x larger using similarly stringent cut-

344 offs. Critically, all of these ARGs were found in phages that are predicted to be temperate or

345 have been engineered to carry ARGs as a marker for selection. With the frequency of

346 carriage in temperate phages being ~1% overall. However, this data is still biased by the

347 majority of temperate phages being isolated on only three bacterial genera. Notably no

348 ARGs were detected on phages of Mycobacterium, which accounts for ~28 % of temperate

349 phages. In comparison, ~2.6% (27/1055) of temperate phages of Streptococcus carry

350 putative ARGs and 50% of phages from Erysipelothrix (1/2). Clearly a much deeper sampling bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

351 of temperate phages from a broader range of hosts is required to get an accurate

352 understanding of the role of phage in the carriage of ARGs. Based on the skewed data

353 available to date, it seems unlikely there will be issues in the isolation of lytic phages for

354 therapeutic use that contain known ARG within their genomes. However, we cannot

355 determine whether these lytic phages cannot spread ARGs via transduction, or through

356 carriage of as-yet uncharacterised ARGs.

357

358 Whilst there is much debate on the presence and importance of ARGs in phage genomes,

359 the role of genes encoding virulence factors is well studied and the process of lysogenic

360 conversion well known (7–10). However, how widespread known virulence genes are in

361 phages is not widely reported. We estimate 1.6% of phages encode at least one putative

362 virulence factor, with the frequency of carriage far higher in temperate phages (5.5%) than

363 lytic phages (0.13%). Again, these overall percentages are skewed by host bias with no

364 known virulence factors detected in Mycobacterium temperate phages (0/1217), in

365 comparison 72% of temperate phages of Shigella (5/7) and 7% (61/846) of Streptococcus

366 contain virulence factors. It is currently impossible to determine if the higher proportion of

367 ARGs and virulence factors in phages of known pathogens is a feature of their biology, or a

368 skew in the database towards phages of clinically relevant isolates.

369

370 Given the biases in the dataset, it is not clear if the general phage patterns we observe (e.g.,

371 jumbo-phages are rarely isolated, more temperate phages on particular hosts, and the

372 carriage of ARGs and virulence genes) are linked to biology or chronic under sampling of

373 phage genomes. We speculate that currently is most likely the latter, which distorts some

374 generalisations about phages. It clear that jumbo-phages are not rare on some hosts and bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

375 putative ARGs are far more abundant on temperate phages. However, far deeper sampling

376 of phage diversity across different hosts is required at an increasing rate.

377

378 Conclusions

379 We have provided a simple method to automate the download of curated set of complete

380 genomes from cultured phage isolates, providing metadata in a format that can be used as a

381 starting point for many common analyses. Analysis of the current data highlights what we

382 know about phage genomes is skewed by the majority of phages having been isolated from

383 a small number of bacterial genera. Furthermore, the rate at which phage genomes are

384 being deposited is decreasing. Whilst understanding of genomic diversity is always

385 influenced by the data available, this is particularly acute for phage genomes with so many

386 phages isolated on smaller number of hosts. To obtain a greater understanding of phage

387 diversity, larger numbers of phages, in particular temperate phages, isolated from a broader

388 range of bacteria need to be sequenced.

389

390

391 Acknowledgments

392

393 Authorship Confirmation Statement

394 Competing Interests.

395 Funding

396 R.C is supported by a scholarship from the Medical Research Foundation National PhD Training

397 Programme in Antimicrobial Resistance Research (MRF-145-0004-TPG-AVISO). A.M, D.J.S, M.J, and

398 J.H were supported by NERC (NE/N019881/1). A.M was supported by MRC

399 (MR/T030062/1and MR/L015080/1) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

400

401 Figure 1

402 Number of complete phage genomes in GenBank over time. Dates were estimated based on date of

403 submission (* for 235 genomes, the date of update was used as no submission date was available).

404 The reference lines showing doubling rates (dashed) begin in 1989, as this is when the number of

405 phage genomes increased beyond the first submission in 1982.

406

407 Figure 2

408 Overall properties of phages. A) Proportion of phages isolated on the top 30 most abundant hosts. B)

409 Distribution of phage genome sizes. C) Proportion of “jumbo-phages” on top 30 hosts for which at

410 least one “jumbo-phage” has been isolated.

411

412 Figure 3

413 Genomic diversity of phages on the top ten most abundant hosts. A) Rarefaction curve of phage

414 species. Species were defined as 95% identity over 95% of genome length. B) Rarefaction curve of

415 phage genera. Genera were defined as 70% identity over 95% of genome length.

416

417 Figure 4

418 Phylogenetic tree of translated terL gene for 313 “jumbo-phages” and their closest relatives. The

419 alignment was produced using MAFFT (36) and tree produced using IqTree using WAG model with

420 1000 bootstrap repeats (37). Pink shaded regions indicate “jumbo-phages”, coloured ring indicates

421 viral genus, and blue bars indicate genome length. Bootstrap values indicated by black circles are

422 shown with a minimum of 70%.

423 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

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571

572 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

573 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made ~5kbavailable under aCC-BY-NC 4.0 International license.

~40kb

~50kb Jumbophage cut-off

~165kb

C A

Streptococcus

Mycobacterium

Escherichia

Vibrio Pooled bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102Salmonella; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is theGordonia author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0Pseudomonas International license.

Staphylococcus Lactococcus

Synechococcus 0 1000 2000 3000 0 2000 4000 6000

Streptococcus

Mycobacterium

Escherichia

Vibrio Pooled Gordonia Salmonella Pseudomonas

Staphylococcus Lactococcus

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Tree scale: 1

Caulobacter phage CcrColossus (JX100810)

Caulobacter phage CcrBL10 (MH588544) Mesorhizobium phage Cp1R7A-A1 (MT188704)

Vibrio phage 1.161.O._10N.261.48.C5 (MG592529) Caulobacter virus Magneto (JX100812) Ruegeria phage vB_RpoS-V10 (MH015255) Caulobacter phage CcrPW (MH588545) Caulobacter phage CcrBL9 (MH588546) Bacteriophage DSS3_MAL1 (MN602268)

Caulobacter phage Ccr2 (KY555143)

Caulobacter virus Karma (JX100811) Roseobacter phage DSS3P8 (KT870145) Caulobacter phage CcrSC (MH588547) Caulobacter phage Ccr5 (KY555144) Caulobacter virus Rogue (JX100814) Sphingobium phage Lacusarx (KY629563) Synechococcus phage S-RIM8 (MK493322) Caulobacter virus Swift (JX100809) Phaeobacter phage MD18 (MT270409) Synechococcus phage S-RIM8 (MK493325)

Synechococcus phage S-RIM8 (MK493324)

Synechococcus phage S-RIM8 (MK493323) Stx2-converting phage Stx2a F578 (LC487997)

Escherichia phage TL-2011a (JQ011316)

Staphylococcus phage Twillingate (MH321491) Planktothrix phage PaV-LD (HQ683709) Methanosarcina virus MetMV (MK170447) Staphylococcus phage Terranova (MH542234) Mycobacterium phage Sheen (KP273225)

Enterococcus phage 156 (LR031359)

Staphylococcus phage S25-3 (AB853330)

Staphylococcus phage SPbeta-like (KT429160)Staphylococcus phage S25-4 (AB853331)

Bacillus phage vB_BceM_WH1 (LC597490)

Pseudoalteromonas phage HM1 (KF302034)

Clostridium phage phi8074-B1 (JQ246028) Bacillus phage SPG24 (AB930182)

Caulobacter phage Ccr29 (KY555145) Caulobacter phage Ccr10 (KY555142)

Caulobacter virus phiCbK (JX163858) Caulobacter phage Ccr34 (KY555147) Caulobacter phage Ccr32 (KY555146)

Caulobacter virus phiCbK (JX100813)

Ruegeria phage vB_RpoP-V13 (MH015256)

Mycobacterium phage PP (AP018486)

Mycobacterium phage Settecandela (MT114163)

Mycobacterium phage Phrappuccino (MK937592)

Escherichia phage Min27 (EU311208)

Stx2-converting phage Stx2a_F451 (AP012532)

Stx2-converting phage Stx2a_F403 (AP012529)

Vibrio phage 1.255.O._10N.286.45.F1 (MG592621)

Vibrio phage 1.244.A._10N.261.54.C3 (MG592609) Bacillus phage BCD7 (JN712910) Staphylococcus phage Machias (MW349128) Staphylococcus phage Madawaska (MW349129) Streptomyces phage MindFlayer (MW291014) Staphylococcus phage MarsHill (MW248466) Streptomyces phage Wipeout (MN484599) Yersinia phage phiR1-37 (AJ972879) Streptomyces phage StarPlatinum (MH576965) Bacillus virus G (JN638751) Bacillus phage vB_BpuM-BpSp (KT895374) Halovirus HGTV-1 (KC292026) Bacillus virus PBS1 (MF360957) Vibrio phage JSF10 (KY883654) Bacillus phage AR9 (KU878088)

Edwardsiella phage pEt-SU (MK689364)

Vibrio phage JSF12 (KY883655) Klebsiella phage N1M2 (MN642089)

Pseudomonas phage OBP (JN627160) Streptomyces phage Wofford (MH576968) Aeromonas phage CF8 (MK774614) Vibrio phage phi 3 (KP280063) Aeromonas phage PS2 (MN453779) Streptomyces phage BoomerJR (MK359351) Aeromonas phage PS1 (MN032614) Streptomyces phage Yaboi (MH727564) Aeromonas phage D6 (MN131137) Aeromonas phage LAh10 (MK838116)

Aeromonas phage D9 (MN159079)

Streptomyces phage Genie2 (MK359332) Aeromonas phage D3 (MN102098) Streptomyces phage Sushi23 (MF358542) Pseudomonas phage EL (AJ697969) Erwinia phage vB_EamM_Joad (MF459647) Streptomyces phage Tribute (MN369743) Erwinia phage vB_EamM_RisingSun (MF459646) Klebsiella phage Miami (MT701590) Streptomyces phage Teutsch (MK460248) Vibrio phage VP4B (KC131130) Streptomyces phage MulchMansion (MT897905) Vibrio phage pTD1 (AP017972) Streptomyces phage EGole (MK494112) Streptomyces phage LilMartin (MT684590) Photobacterium phage PDCC-1 (MN562221) Vibrio phage 2 TSL-2019 (MK368614)

Vibrio phage Aphrodite1 (MG720308)

Streptomyces phage Blueeyedbeauty (MH536814) Vibrio phage USC-1 (MK905543) Streptomyces phage Daubenski (MN444876) Streptomyces phage Bmoc (MT310865) Vibrio phage 5 TSL-2019 (MK358448) Salmonella phage SPFM9 (LR535907)

Salmonella phage SPFM1 (LR535901)

Streptomyces phage Annadreamy (MH536811) Salmonella phage SPFM12 (LR535911) Streptomyces phage Beuffert (MT952852) Salmonella phage SPFM6 (LR535905)

Salmonella phage SPFM10 (LR535908)

Salmonella phage SPFM15 (LR535913) Streptomyces phage Limpid (MN369754) Salmonella phage SPFM14 (LR535912)

Salmonella phage SPFM5 (LR535903) Streptomyces phage Faust (MT684598) Salmonella phage SPFM7 (LR535904)

RhodococcusStreptomyces phage Weasels2 phage Moab(KX774321) (MT114162) Salmonella phage SPFM8 (LR535906) Rhodococcus phage NiceHouse (MT521992) Salmonella phage SPFM20 (LR535917)

Vibrio phage pVa-21 (KY499642) Methanocaldococcus fervens tailed virus 1 (MT711370) Cronobacter phage CR5 (JX094500) Brevibacillus phage SecTim467 (KT151957) Brevibacillus phage Jenst (KT151955) Erwinia phage vB_EamM_Phobos (KX397372) Erwinia phage Derbicus (MK514282)

Erwinia phage vB_EamM_EarlPhillipIV (KX397367) Bacillus phage EZ-2018a (CP027117) Erwinia phage Wellington (MH426724)

Stenotrophomonas phage vB_SmaS_DLP_3 (MT110073) Erwinia phage vB_EamM_Kwan (KX397369)

Erwinia phage vB_EamM_ChrisDB (KX397366)

Erwinia phage vB_EamM_Caitlin (KX397365)

Vibrio phage vB_VcaS_HC (MK559459) Erwinia phage pEa_SNUABM_43 (MW384823) Vibrio phage Ares1 (MG720309) Vibrio phage 1 (JF713456) Salmonella phage pSal-SNUABM-04 (MT710307) Bacillus phage vB_BceM-HSE3 (MF418016) Erwinia phage vB_EamM_Parshik (KX397371)

Erwinia phage Machina (KX397370)

Erwinia phage vB_EamM_Huxley (KX397368)

Synechococcus phageBacillus S-CBS4 phage (HQ698895) SP-15 (KT624200) Erwinia phage vB_EamM_Asesino (KX397364) Gordonia phage Tredge (MK494098) Erwinia phage vB_EamM_Alexandra (MH248138) Erwinia phage vB_EamM_Stratton (KX397373) Erwinia phage phiEaH2 (JX316028)

Salmonella phage SPAsTU (MH221129) Dickeya phage vB_DsoM_AD1 (MH460463) uncultured phage (LR745208) Salmonella phage JN03 (MT799840) Escherichia phage vB_EcoM_EC001 (MN445185) Erwinia phageErwinia vB_EamM_Yoloswag phage vB_EamM_Y3 (KY448244) (KY984068) Enterobacteria phage SEGD1 (KU726251)

Erwinia phage pEa_SNUABM_5 (MW366843) Salmonella phage SPFM17 (LR535914)

Salmonella phage SPFM19 (LR535916) Dickeya phage vB_DsoM_JA11 (MH389777) Salmonella phage SPFM4 (LR535902) Dickeya phage vB_DsoM_JA33 (MH460462) Proteus phage 7 (MT679221) Dickeya phage vB_DsoM_JA13 (MH460460) Salmonella phage SPFM16 (LR535915)

Dickeya phage vB_DsoM_JA29 (MH460461) Salmonella phage SPFM13 (LR535910)

Salmonella phage SPFM2 (LR535921)

Salmonella phage SPFM3 (LR535920)

Pseudomonas phage Lu11 (JQ768459) Salmonella phage SPFM11 (LR535909)

BurkholderiaPseudomonas phage BcepSaruman phage PaBGPhage (MK552140) (KF147891) NCTB (LT598654) Salmonella phage SPFM21 (LR535919) Salmonella phage SPFM22 (LR535918) Burkholderia phage BcepSauron (MK552141) 600 kb Salmonella phage SPN3US (JN641803) Achromobacter phage Motura (MN094788) Vibrio phage BONAISHI (MH595538) 500 kb Vibrio phage vB_VcorM_GR11A (MT366761) Vibrio phage vB_pir03 (MT811961)

Vibrio phage vB_VcorM_GR7B (MT366760) Vibrio phage vB_VmeM-Yong XC32 (MK308675) 400 kb Vibrio phage vB_VmeM-Yong MS31 (MK308676) 300 kb Vibrio phage vB_VmeM-Yong MS32 (MK308677) Thermus phage TMA (AP011617) Vibrio phage vB_VmeM-Yong XC31 (MK308674) 200 kb MicrocystisCyanophage virus Ma-LMM01 S-TIM5 (AB231700) (JQ245707) Ralstonia phage RP31 (AP017925) Microcystis phage MaMV-DC (KF356199) Ralstonia phage RP12 (AP017924) 100 kb Ralstonia phage RSF1 (AP014927)

Ralstonia phage RSL2 (AP014693)

Xanthomonas phage Xoo-sp14 (MT939492)

Proteus phage 10 (MT661596)

Salmonella phage vB_SalM_SA002 (MN445183)

Erwinia phage vB_EamM_Special G (KU886222)

Erwinia phage vB_EamM_MadMel (MG655269)

Erwinia phage vB_EamM_Deimos-Minion (KU886225)

Erwinia phage vB_EamM_Mortimer (MG655270)

Erwinia phage Rebecca (MK514281)

Erwinia phage vB_EamM_Simmy50 (KU886223)

Erwinia phage vB_EamM_Bosolaphorus (MG655267)

Erwinia phage vB_EamM_Desertfox (MG655268)

Erwinia phage vB_EamM_RAY (KU886224)

Erwinia phage Ea35-70 (KF806589)

Escherichia phage vB_EcoM_Goslar (MK327938)

Serratia phage KpHz_2 (MN871445)

Serratia phage KpGz_1 (MN871444)

Serratia phage PCH45 (MN334766) Synechococcus phage ACG-2014f (KJ019037) Erwinia phage PhiEaH1 (KF623294) Synechococcus phage ACG-2014f (KJ019053) Serratia phage Moabite (MK994515) Synechococcus phage ACG-2014f (KJ019111) Serratia phage vB_SmaM_Yaphecito (MW021758) Synechococcus phage ACG-2014f (KJ019148) Serratia phage 2050HW (MF285618)

Synechococcus phage ACG-2014f (KJ019107) Serratia phage KpLz_1_41 (MN871447)

Synechococcus phage ACG-2014f (KJ019101) Serratia phage KpYy_2_45 (MN871451)

Synechococcus phage ACG-2014f (KJ019123) Serratia phage KpLz-2_45 (MN871446)

Synechococcus phage ACG-2014f (KJ019155) Serratia phage KpXa_1 (MN871449)

Synechococcus phage ACG-2014f (KJ019147) Serratia phage KpZh_LPS_45 (MN871453) Serratia phage KpZh_1 (MN871452) Synechococcus phage ACG-2014f (KJ019150) Serratia phage KpSz_1 (MN871448) Synechococcus phage ACG-2014f (KJ019159) Pseudomonas phage Noxifer (MF063068) Synechococcus phage ACG-2014f (KJ019152) Pseudomonas phage Psa21 (MK552327) Synechococcus phage ACG-2014f (KJ019066) Pseudomonas phage vB_PaeM_PS119XW (MN103543)

Synechococcus phage ACG-2014f (KJ019143) Pseudomonas phage PA1C (MK599315) Synechococcus phage ACG-2014f_Syn7803C7 (KJ019052) Pseudomonas phage PhiPA3 (HQ630627)

Synechococcus phage ACG-2014f (KJ019093) Pseudomonas phage Phabio (MF042360)

Synechococcus phage ACG-2014f (KJ019092) Pseudomonas phage 201phi2-1 (EU197055)

Synechococcus phage ACG-2014f (KJ019149) Pseudomonas phage vB_PaeM_kmuB (MW057919) Pseudomonas phage KTN4 (KU521356) Synechococcus phage ACG-2014f (KJ019085) Pseudomonas phage PaZh_1 (MN871489) Synechococcus phage ACG-2014f (KJ019144) Pseudomonas phage PaSz-1_45_270k (MN871480) Synechococcus phage ACG-2014f (KJ019105) Pseudomonas phage SL2 (MF805716) Synechococcus phage ACG-2014f (KJ019059) Pseudomonas virus phiKZ (AF399011) Synechococcus phage ACG-2014f_Syn7803C8 (KJ019058) Pseudomonas phage PA7 (JX233784)

Synechococcus phage ACG-2014f (KJ019095) Pseudomonas phage PA02 (AP019418)

Synechococcus phage ACG-2014f (KJ019142) Pseudomonas phage fnug (MT133560)

Synechococcus phage ACG-2014f (KJ019099) Streptococcus phage Javan584 (MK448990)

Synechococcus phage ACG-2014f (KJ019086) Streptococcus phage Javan577 (MK448812) Arthrobacter phage Sonali (MK411746) Synechococcus phage ACG-2014f (KJ019145) Roseobacter phage CRP-3 (MK613345) Synechococcus phage ACG-2014f (KJ019098) Exiguobacterium phage vB_EalM-132 (MH884511) Synechococcus phage ACG-2014f (KJ019106) Pseudomonas phage 22PfluR64PP (MH179472) Synechococcus phage ACG-2014f (KJ019146) Bacillus phage 0305phi8-36 (EF583821) Synechococcus phage ACG-2014f (KJ019102) Pseudoalteromonas phage PHS21 (KY379511)

Synechococcus phage ACG-2014f (KJ019100) Pseudoalteromonas phage SL25 (MF370965)

Synechococcus phage ACG-2014f (KJ019090) Pseudoalteromonas phage AL (MT002875)

Synechococcus phage ACG-2014f (KJ019096) Pseudoalteromonas phage PHS3 (KX912252)

Synechococcus phage ACG-2014f (KJ019141) Pseudomonas phage vB_PaeS_SCUT-S4 (MK165658)

Synechococcus phage ACG-2014f (KJ019045) Stenotrophomonas phage vB_SmaS-DLP_2 (KR537871) Pseudomonas phage UFV-P2 (JX863101) Synechococcus phage ACG-2014f (KJ019097) Pseudomonas phage PaYy-2 (MH725810) Synechococcus phage ACG-2014f_Syn7803US26 (KJ019091) Pseudomonas phage Pa-U (MN871470) Synechococcus phage ACG-2014f (KJ019151) Pseudomonas phage PaYy-2 (MN871488) Synechococcus phage S-CAM7 (KU686212) Pseudomonas phage SRT6 (MH370478) Synechococcus phage S-CAM7 (MT586120) Sphingomonas phage PAU (JQ362498)

Synechococcus phage S-CAM7 (KU686213) Tenacibaculum phage pT24 (LC168164)

Synechococcus phage S-SSM7 (GU071098) Tenacibaculum phage PTm5 (AP019525) Cyanophage S-SSM6a (HQ317391) Tenacibaculum phage PTm1 (AP019524)

Synechococcus phage ACG-2014j (KJ019069) uncultured phage (LR756500)

Synechococcus phage S-CAM4 (KU686202) uncultured phage (LR756504) uncultured phage (LR756502) Synechococcus phage S-CAM4 (KU686201) uncultured phage (LR745206) Synechococcus phage ACG-2014e (KJ019156) Acinetobacter phage vB_AbaM_ME3 (KU935715) Synechococcus phage S-CAM4 (KU686200) Halocynthia phage JM-2012 (JQ340088) Synechococcus phage S-RSM4 (FM207411) Prevotella phage Lak-A2 (MK250019) Synechococcus phage S-H9-1 (MW117966) Prevotella phage Lak-C1 (MK250029)

Synechococcus phage S-WAM2 (KU686211) Prevotella phage Lak-A1 (MK250016)

Synechococcus phage S-CAM1 (KU686193) Prevotella phage Lak-A1 (MK250017)

Synechococcus phage S-CAM1 (KU686196) Prevotella phage Lak-A1 (MK250018)

Synechococcus phage S-CAM1 (KU686195) Prevotella phage Lak-A1 (MK250015)

Synechococcus phage S-CAM1 (KU686194) Prevotella phage Lak-B2 (MK250021)

Synechococcus phage S-PM2 (AJ630128) Prevotella phage Lak-B7 (MK250026) Prevotella phage Lak-B4 (MK250023) Synechococcus phage S-PM2 (LN828717) Cyanophage S-RIM32 (KU594606) Prevotella phage Lak-B5 (MK250024)

Prevotella phage Lak-B6 (MK250025) Prochlorococcus phage P-HM1 (GU071101) Prevotella phage Lak-B3 (MK250022)

Prochlorococcus phage MED4-213 (HQ634174) Prevotella phage Lak-B8 (MK250027)

Prochlorococcus phage P-HM2 (GU075905) Prevotella phage Lak-B9 (MK250028)

Synechococcus phage S-SM2 (GU071095) Prevotella phage Lak-B1 (MK250020)

Synechococcus phage Bellamy (MF351863) Gordonia phage BrutonGaster (MK524501)

Roseobacter phage CRP-7 (MK613349) Prochlorococcus phage P-TIM68 (KM359505) Bacillus phage BM5 (KT995479)

uncultured phage (LR756511) SynechococcusSynechococcus phage SynMITS9220M01 phage S-B43 (MT408532) (MN018232) Phage DP SC_6_H4_2017 (MT121964) Synechococcus phage S-B05 (MK799832) Vibrio phage 1.205.O._10N.222.51.A7 (MG592575)

Synechococcus phage S-SCSM1 (MK867354) Aeromonas phage phiARM81mr (KT898134)

Synechococcus phage S-CAM9 (KU686204) uncultured phage (LR756508)

Synechococcus phage S-CAM9 (KU686206) Flavobacterium phage vB_FspM_immuto_13-6C (MW353177)

Synechococcus phage S-CAM9 (KU686205) Flavobacterium phage vB_FspM_immuto_3-5A (MW353176)

Synechococcus virus S-PRM1 (MH629685) Flavobacterium phage vB_FspM_immuto_2-6A (MW353175)

Synechococcus phage S-SKS1 (HQ633071) Xanthomonas phage Xoo-sp13 (MN047793) Xanthomonas phage XacN1 (AP018399) Prochlorococcus phage P-SSM2 (AY939844) Salicola phage SCTP-2 (MF360958) Prochlorococcus phage P-SSM2 (GU071092) Agrobacterium phage Atu_ph07 (MF403008) Prochlorococcus phage P-SSM5 (HQ632825) Marine virus AG-345-F15 (MH319749) Pseudomonas phage vB_PaeM_PA5oct (MK797984)

Marine virus AG-345-L05 (MH319754) Pseudomonas phage vB_PaeM_MIJ3 (LR588166) Vibrio phage 2.275.O._10N.286.54.E11 (MG592671) Synechococcus phage S-T4 (MH412654) Pectinobacterium phage vB_PcaM_CBB (KU574722)

ProchlorococcusCyanophage phage P-SSM4 P-RSM3 (AY940168) (HQ634176) Cronobacter phage vB_CsaM_GAP32 (JN882285) Yersinia phage fHe-Yen9-03 (LT960552)

Yersinia phage fHe-Yen9-04 (LT960551) Cyanophage P-RSM1 (HQ634175) Prochlorococcus phage P-SSM3 (HQ337021) Yersinia phage fHe-Yen9-04 (LR596615)

Erwinia phage pEa_SNUABM_12 (MT939486) SynechococcusCyanophage phage S-RIM2 S-RIM12 (KX349248) (KX349320) Erwinia phage pEa_SNUABM_50 (MT939488) Cyanophage S-RIM50 (KU594605) Erwinia phage pEa_SNUABM_47 (MT939487)

Serratia phage BF (KY630187) Cyanophage Syn2 (HQ634190) Synechococcus phage Syn19 (GU071106) Salmonella phage 7t3 (MK773491)

Salmonella phage SE_PL (MT001829)

Salmonella phage Munch (MK268344) Prochlorococcus phage P-SSM7 (GU071103) Cyanophage S-TIM4 (MH512890) Klebsiella phage vB_KleM_RaK2 (JQ513383) Prochlorococcus phage P-RSM4 (GU071099) Klebsiella phage Muenster (MT708547)

Klebsiella phage K64-1 (AB897757)

Synechococcus phage S-SSM5 (GU071097) Escherichia phage CMSTMSU (MH494197)

Synechococcus phage S-SM1 (GU071094) Escherichia virus TH47 (MT446416)

Synechococcus phage Syn30 (HQ634189) Escherichia phage Ecwhy_1 (MH791411)

Synechococcus phage S-E7 (MH920640) Escherichia phage 121Q (KM507819)

Escherichia phage SP27 (LC494302)

Synechococcus phage ACG-2014c (KJ019027) Escherichia phage vB_EcoM_phAPEC6 (MK817115) Escherichia phage vB_Eco_slurp01 (LT603033) Synechococcus phage ACG-2014c (KJ019128) Escherichia phage vB_EcoM_G17 (MK327931) Synechococcus phage ACG-2014c (KJ019064) Escherichia phage UB (MH383160)

SynechococcusSynechococcus phage ACG-2014c phage S-H35 (KJ019063) (KY945241) Escherichia phage PBECO4 (KC295538) Cyanophage S-RIM4 (MK493321) Synechococcus phage S-B64 (MH107246) Vibrio phage 1.121.O._10N.286.46.C4 (MG592495) Vibrio phage phiVa1 (MK387337) Cyanophage Syn10 (HQ634191) Campylobacter phage CP20 (MK408758) Synechococcus phage syn9 (DQ149023) Campylobacter virus CPt10 (FN667789)

Agrobacterium phage Atu_ph04 (MF403007)

Sinorhizobium phage phiM9 (KP881232) Synechococcus phage S-WAM1 (KU686210) Vibrio phage 1.081.O._10N.286.52.C2 (MG592456)

Vibrio phage V09 (MT135026) Synechococcus phage ACG-2014d (KJ019118) Vibrio phage KVP40 (AY283928) Synechococcus phage ACG-2014d (KJ019140) Vibriophage phi-pp2 (JN849462)

Synechococcus phage ACG-2014d (KJ019165) Vibrio phage VH1_2019 (MN794232)

Synechococcus phage ACG-2014d (KJ019162) Vibrio phage V07 (MT135025) Vibrio phage V05 (MT135024) Synechococcus phage ACG-2014d (KJ019164) Cyanophage S-SSM6b (HQ316603) Vibrio phage nt-1 (HQ317393) Synechococcus phage ACG-2014d (KJ019136) Vibrio phage ValKK3 (KP671755) Synechococcus phageCyanophage S-SSM4 (HQ316583) S-RIM14 (KX349301) Vibrio phage vB_ValM_R11Z (MT612989) Cyanophage S-RIM14 (KX349306) Vibrio phage phi-Grn1 (KT919972) Cyanophage S-RIM14 (KX349305) Vibrio phage phiVa3 (MK568540)

Cyanophage S-RIM14 (KX349304) Vibrio phage vB_ValM_R10Z (MT612988) Cyanophage S-RIM14 (KX349302) Vibrio phage phi-ST2 (KT919973) Vibrio phage VH7D (KC131129) Cyanophage S-RIM14 (KX349303) Cronobacter phage S13 (KC954775)

Aeromonas phage Ahp1 (MT161459)

Aeromonas phage phiAS5 (HM452126)

Aeromonas phage Ah1 (MG250483)

Synechococcus phage S-ShM2 (GU071096) Aeromonas virus Aeh1 (AY266303)

Synechococcus phage S-H38 (MW117965) Aeromonas phage AsFcp_2 (MH791401)

Synechococcus phage S-B68 (MK016664) Aeromonas phage PX29 (GU396103)

Aeromonas phage AsFcp_1 (MN871440) Synechococcus phage S-N03 (MT162466) Aeromonas phage AsFcp_4 (MH791407)

Vibrio phage vB_VmeM-32 (KU160494) Pelagibacter phage HTVC008M (KC465899) Synechococcus phage B3 (MN695334) Aeromonas phage ZPAH1 (MW272540) Aeromonas phage Aswh_1 (MH791414)

Aeromonas phage 65.2 (KY290955) Synechococcus phage B23 (MN695335) Aeromonas virus 65 (GU459069) Pelagibacter phage Mosig EXVC030M (MT647606) Aeromonas phage AS-zj (MF448340) Salmonella phage SS3 (MK972698) Aeromonas phage CC2 (JX123262) Salmonella phage SS9 (MK972699) Aeromonas phage AP1 (MT713136) Aeromonas phage AS-sw (MF498775)

Salmonella phage moki (MT074478) Aeromonas phage Asswx_1 (MH791398)

Aeromonas phage Aszh-1 (MN871442) Ochrobactrum phage vB_OspM_OC (MT028491) Aeromonas phage AsSzw2 (MN871441) Aeromonas phage Assk (MH791404) Salmonella phageSalmonella dinky phage(MT074475) 38 (KR296692) Salmonella phage Chennai (MN953776) Aeromonas phage AS-szw (MF498773)

Stenotrophomonas phage vB_SmaS-DLP_6 (KU682439) Cronobacter phage vB_CsaM_GAP161 (JN882287)

Enterobacter phage EC-F1 (MN508623)

Cronobacter phage vB_CsaM_SemperBestia (MW021756)

Salmonella phage barely (MT074477) Aeromonas phage asfd_1h (MN871507) Serratia phage phiMAM1 (JX878496) Aeromonas virus 31 (AY962392) Aeromonas phage Riv-10 (KY290957) Serratia phage 2050H1 (MF285619) Serratia phage KNP4 (KX452697)Vibrio phage YC (MH375644) Aeromonas phage SW69-9 (KY290958)

Aeromonas phage L9-6 (KY290956)

Escherichia phage vB_EcoM_PHB13 (MK573636)

Escherichia phage kvi (MN850615)

Escherichia phage ECD7 (KY683735)

Escherichia virus Ec_Makalu_003 (MN882349)

Escherichia virus Ec_Makalu_002 (MN709127)

Vibrio phage VAP7 (MK795384) Providencia phage PSTCR6 (MW057858)

Providencia phage PSTRCR_127 (MW358927)

Morganella phage vB_MmoM_MP1 (KX078569)

Proteus phage phiP4-3 (MG696114)

Proteus phage PM2 (MF001355)

Proteus phage vB_PmiM_Pm5461 (KP890823)

Acinetobacter phage AbTZA1 (MK278860) Serratia phage vB_SmaA_3M (MH929319) Acinetobacter phage Melin (MW388003) Acinetobacter phage Morttis (MW388004)

Acinetobacter phage Mokit (MW388002)

Serratia phage Muldoon (MN095771)

Serratia phage PS2 (KJ025957)

irbce phage CF1 ERZ-2017Citrobacter (MG250484)

Citrobacter phage Moon (KM236240)

Klebsiella phage vB_KaeM_KaAlpha (MN013084)

Vibrio phage vB_VcorM_GR28A (MT366762) Vibrio phage vB_VchM_Kuja (MN718199) Pseudomonas phage pf16 (KU873925)

Acinetobacter phage SH-Ab 15599 (MH517022) Acidovorax phage ACP17 (KY979132) Stenotrophomonas phage YB07 (MK580972)

Stenotrophomonas phage Mendera (MN098328) Phage NC-G (MK310184)

Stenotrophomonas phage Moby (MN095772)Sinorhizobium phage phiM12 (KF381361) Escherichia virus TH40 (MT446411)

Escherichia virus TH55 (MT446421) Stenotrophomonas phage IME-SM1 (KR560069) Sinorhizobium phageSinorhizobium phiM19 (KR052481) phage phiM7 (KR052480) Sinorhizobium phage phiN3 (KR052482)

Escherichia virus TH54 (MT446420)

Escherichia phage UPEC01 (MW233709) Escherichia phage JS10 (EU863409)

Escherichia phage ST0 (MF044457)

Klebsiella phage EI (MN106245)

Escherichia phage HX01 (JX536493)

Klebsiella phage Marfa (MN044033) Serratia phage CBH8 (MF036691)

Escherichia phage vB_EcoM_VR7 (HM563683)

Escherichia phage EcS1 (LC371242)

Escherichia phage vB_EcoM_VR26 (KP007362) Klebsiella phage KP179 (MH729874)

Klebsiella phage vB_Kpn_F48 (MG746602) Enterobacter phage CC31 (GU323318)

Klebsiella phage vB_Kpn_P545 (MN781108)

Escherichia phage vB_EcoM_NBG1 (MH243438) Klebsiella phage Metamorpho (MT701588)

Escherichia phage vB_EcoM_G2285 (MK327933) Klebsiella phage vB_KpnM_Potts1 (MN013081) Enterobacter phage prasa_myo (MN617835)

Pectobacterium bacteriophage PM2 (KF835987)

Klebsiella phage vB_KpnM_KpV477 (KX258185) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Write genomes that pass filters Download phage records from Produce MASH index for easy to new file (this is the final Genbank using "PHG" tag and comparison of new sequences database), and write individual length cutoffs against known genomes records to /GenomesDB/

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Exclude genomes using manually curated list of Produce annotation files for Produce annotation files for accession numbers for known vConTACT2 IToL erroneous genomes A

C bioRxiv preprint doi: https://doi.org/10.1101/2021.05.01.442102; this version posted May 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Lifestyle Lytic Temperate

Features ARG

Virulence Factor

Jumbophage