BACKGROUND
the value of domestic ruminants  comes from their ability to convert, by means of rumen fermentation, low-quality forages into high quality, high protein products  suitable for human consumption  <cit> .
the rumen, the first section of the ruminant stomach, contains symbiotic microorganisms that breakdown ingested food.
these microorganisms include eukarya, bacteria, archaea <cit> , and large numbers  of virus including bacteriophage  <cit> .
the entire bacteriophage or 'phage’ population in a sample is termed a virome.
massively parallel sequencing  is a technology which allows the generation of several million dna sequences in parallel.
mps has been used to investigate the bacterial and archaeal populations of the bovine  rumen  <cit> .
however, mps studies on the rumen virome are scant.
one study  <cit>  sequenced  <dig> mbp of viral dna from the rumen of three animals, one lactating, one non lactating and one culled animal.
the mps study  <cit>  and earlier studies such as  <cit> , both suggest that there are large amounts of variation in the bovine rumen virome.
however some of the variation may have been due to external sources .
since the recent rumen virome study  <cit> , there have been technological advances that have allowed the volume of sequence data to increase by an order of magnitude.
these advances now enable the sequencing of viromes from a greater numbers of animals, resulting in the ability to gain an insight into natural variation between animals.
mps virome studies have made it possible to study viral genomes even when the host bacterium cannot be cultured.
however, there are significant challenges involving analysis of mps virome data  <cit> .
tools for the analysis of virome samples  <cit>  are becoming more readily available allowing estimates of virome characteristics such as species richness.
the combination of published analysis techniques, along with the increased ability to generate data has allowed the field to switch from descriptive analyses to quantitative investigations of how various factors  affect the virome  <cit> .
to access the amount of variation in the rumen virome we extracted virus like dna from the rumen fluid of  <dig> lactating dairy cattle.
twelve of the  <dig> cows were housed together, fed the same diet, had a shared location and were sampled on the same day, allowing us to investigate the magnitude of natural variation in the rumen virome.
RESULTS
assembly and annotation
we sequenced virus like particles from the rumen fluid of  <dig> lactating australian holstein dairy cattle on the hiseq <dig>  each library contained between  <dig> and  <dig> million read pairs .
after a combined assembly of all samples,  <dig> contigs were greater than  <dig> kbp in length.
these contigs were manually inspected for even coverage and annotated .
three of the contigs have all the genes necessary for a complete functional phage genome .
we mapped our sequence reads back to the complete set of contigs.
while up to  <dig> % of reads mapped to the whole assembly, only  <dig> % of reads mapped to contigs greater than  <dig> kbp .
this would suggest that although we have assembled up to 57% of the bovine rumen virome, most of what has been assembled is still highly fragmented, and contained in small contigs.
t
+
+
+
b
d
*average of all samples from this study.
ccannulated animal.
tamount of sequence remaining after removal of poor quality bases.
+multiple samples from study combined.
bbest model as assessed by the catchall program.
dbest model that has been corrected using the assumption of overrepresented singletons.
five of the  <dig> largest contigs were observed in all samples , with all  <dig> observed in the sample from animal  <dig> .
despite over a third of the most common contigs being represented in all samples, the most abundant contig only accounted for  <dig> % of sequence reads .
there was a strong correlation between the sequencing depth of the sample and the percentage of reads that mapped back to the assembly , partly reflecting the fact that the contigs were assembled from the same data.
as sequence depth has a strong impact on contig size, it is likely that we have only assembled genomes from the most dominant phage species in the bovine rumen.
of the  <dig> putative genes in the  <dig> largest contigs,  <dig> % were assigned an unknown function because all of the significant blast hits were themselves unannotated.
genes involved with either dna replication or repair consisted  <dig> % of putative genes,  <dig> % of putative genes were structural and  <dig> % appear to be involved in lysis.
completely novel  putative genes made up an additional  <dig> %.
these novel and unannotated genes may encode unknown functions including those involved in cell lysis or lytogenic reproduction of the phage.
phage may be integrated into the genomes of their host prokraryotes.
to look for evidence of this we aligned sequence data from the microbiomes of the same samples from  <cit>  to the  <dig> largest contigs.
all of the largest contigs were observed in one or more microbiome samples.
this could be explained by viral contamination of the microbiome.
such contamination would result in the same proportions of viral contigs in the microbiome as are observed in the virome.
therefore the proportions of reads that map to the viral contigs in the microbiome were compared to the virome.
the abundance of two contigs, e and m, were significantly higher in the microbiome than expected under the viral contamination model .
this is evidence that these two phage are integrating into the prokaryote genomes.
however this hypothesis that cannot be validated until the host genomes are sequenced or the host and phage and isolated and cultured.
additionally, contigs a, b, c, e, f, g and n show homology to genes involved in lysis, suggesting that they may be lytic, however this is again speculation until the phage are isolated.
variation in contig abundance between animals
the twelve co-habited animals’ viromes appear to be dominated by contigs d, e and j, while animal 2202’s virome shows a much more even coverage between contigs .
however these contigs only account of < <dig> % of the viromes of the co-habited animals.
there also appears to be a heterogeneity in the distributions of contigs between animals .
contig h has a high abundance in cow  <dig> , but is almost below detectable limits in the deepest sequenced animal  and is undetectable in cow  <dig> .
the evenness observed in the virome of animal  <dig> possibly hints that the rumen viral community is not dominated by a single species, but has a large number of taxa with a reasonably even distribution.
this evenness of distribution was likely not observed in our other samples due to their limited sequence depth resulting in many unassembled species.
to assess the distribution on an independent set of contigs we aligned our reads to contigs assembled from the independent set of animals from  <cit> .
between  <dig> % and  <dig> % of our reads aligned to the contigs.
the distribution of contigs was much more even when these independent contigs were used .
a principle component analysis did not separate the independently housed animal from the co-house animal, although as less than 1% of the reads from any virome aligned, this should be interpreted with caution.
the evenness of the phage population concurs with the findings of  <cit>  that even the most abundant phage species represent only a small proportion of the rumen virome.
our finding that the most common of phage contigs only represent  <dig> % of the virome is consistent with this.
the higher degree of similarity between the viromes of the  <dig> co-habited animals and animal  <dig> may suggest that there is transfer of viral species between the hosts within a herd, although  <dig> was also cannulated, this confounding effect is not possible to resolve without further virome data.
still the variation between co-habited animals  illustrates that any transfer between animals does not maintain a completely homogeneous herd.
whether the within herd variation is related to traits that are related to rumen microbiome functions, such as methane emission levels or bloat susceptibility is yet to be seen, however this dataset shows that there is natural within herd virome variation which should be investigated.
future studies may explore the relationship between this variation and host traits such as disease susceptibility and methane emissions.
species richness
based on our sequence data, it is clear that estimations of rumen phage diversity using electron microscopy of less than  <dig> species per host summerised in  <cit>  are dramatic underestimations, and the true number of species likely lies closer to the estimate in  <cit>  of between  <dig> and  <dig> thousand.
to estimate the number of species present in each of our samples, we used catchall  <cit> .
the estimated total number of species were between  <dig>  and  <dig>  million per sample .
over estimations of richness can be caused by spurious singletons, and the biological and technical causes for these have been outlined in  <cit> .
to account for spurious singletons we also used a model which corrects for such artefacts.
this model reported between  <dig>  and  <dig> , <dig>  species per sample.
the divergence between these results and those reported in  <cit>  is not surprising given that the authors of catchall consistently report richness higher than phaccs  <cit> , and claim that phaccs underestimates richness.
our assembly contains  <dig> mbp of assembled sequence, if we assume an average phage genome size of  <dig> kbp this results in approximately  <dig> genomes  worth of sequence in our assembly.
given that we have only assembled half of the sequence in our samples, and the assembled half would be the less diverse than the unassembled half, approximately  <dig> thousand genomes could be expected as a lower bound for number of species estimations.
this rough calculation concurs with the catchall discounted model estimate for cow  <dig>  and together they tend to suggest that previous assessments of rumen phage richness are indeed underestimations.
in any case, it is clear that there is a large amount of variation in the species that make up the b. taurus rumen virome.
also the large range of estimates, even under the reduced model, suggests that the viromes of some animals are much more species rich than others.
this could be an indication of a more diverse prokaryote population in some animals than others.
this large range should be considered in future experimental designs, as deeper sequencing of particularly diverse samples will be needed to capture a snapshot of the viral community.
comparison to other viromes
there are a number of host associated and non host associated viromes sequenced to date.
to compare these virome sequence profiles to ours we obtained reads from publically available datasets derived from published studies.
here we have only isolated viral dna , therefore we were unable to compare our results to studies such as that by  <cit> , which isolated rna viruses.
we selected four different types of viromes for comparison with our data.
the viromes included samples from cattle rumen, human saliva, swine faeces and non host-associated environments  <cit> .
the reads from the published viromes were aligned to our contigs.
the other bovine rumen viromes had only a small percentage  of reads that mapped to our contigs.
when we compared our reads to reads in the virome obtained from the rumen fluid of cattle as previously reported  <cit>  only 2-5% of our reads mapped .
this would suggest that there are substantial differences between the viromes observed by  <cit>  and ourselves.
these differences could be a result of the phage isolation technique  <cit> , or possibly the sequencing technology used.
it is also possible that the differences were due to real variations, as there was large amounts of variation observed within our samples and it could be expected that cows from other locations and on different diets might be even more distinct.
+multiple samples from study combined.
*average of all samples from this study.
we then compared our reads to the non-bovine viromes.
the swine faeces virome  <cit> , the human saliva virome  <cit>  and aquaculture pond derived virome  <cit>  did not have any reads mapping to our contigs .
we then attempted to map our reads to the  <dig> reads of the above mentioned databases.
less than  <dig> % of our reads mapped to the swine faeces database, while less than  <dig> % of our reads mapped to the human saliva or pond viromes .
there is an observable cline in the amount of our reads that map to these databases as they become less similar to our sample type, from highest from our own assembly, followed by the same sample type , then an alternative species digestive tract , then non gut derived viromes.
this cline in reads that align to contigs from different habitats may be a result of the similar prokaryote populations within gut samples.
both swine faeces and bovine rumen are dominated by firmicutes and bacteroidetes phyla  <cit> , while the salivary microbiome is dominated by proteobacteria  <cit> .
hence the viral species in gut samples likely prey mostly on firmicutes and bacteroidetes, which would result in more similarities between the gut derived viromes then between the rumen and non-gut derived viromes.
functional characteristics of the rumen virome
we also investigated the hypothesis that the functional characteristics of the bovine rumen virome were conserved between animals despite the large amount of sequence variation observed.
we assigned out reads to kegg second level pathways.
of the assigned second level kegg pathways, nucleotide metabolism and replication and repair combined made up 57% of virome assignments.
this is in contrast to the prokaryote metagenomes of rumen fluid from animals from the same location  <cit>  .
evenness of second level kegg pathways was significantly lower in the phage sequences compared to the microbe data .
this unevenness in the phage dataset is likely due to the lack of genes coding for functions such as carbohydrate metabolism, which the phage relies on the host cell to provide.
we then compared the functional characteristics of our sequences to other viromes.
a principle component analysis revealed that all viromes clustered, and all microbial metagenomes clustered based on assignments to kegg second level pathways .
these analyses reveal how the virome of a sample is functionally distinct from the microbial metagenome, and that the functional characteristics of viromes are more similar to each other than to microbial metagenomes, even when the viromes are taken from different sample types .
this is likely a reflection of the limited number of annotatable functions encoded for in the phage metagenome.
interestingly the functions coded for in our viromes are not as variable as that observed in human faeces  <cit> .
some of the human faeces variation may be caused by the small number of reads which could be used in the kegg analysis due to the limited percentage of alignments and the starting data volume.
n
*average of all samples from this study.
ccannulated animal.
nnumber of second level kegg categories observed in sample’s data.
h’ – shannon diversity index.
e – evenness .
+multiple samples from study combined.
the relatively narrow functional characteristics observed in the rumen fluid virome may be in part due to the lack of homology to annotated genes in the databases used for annotation .
the potential of novel bacteriophage derived genes to control the bacterial populations of the host will likely be of interest in both a biomedical sense  and, in the case of livestock, reducing enteric methane emissions from the rumen.
rumen virome importance
domestic ruminants are important production animals to people worldwide.
their ability to use microbial fermentation to provide energy for food , fibre  and power  has seen their numbers soar to  <dig>  billion  <cit> .
because of this, researchers have long been interested in the rumen microbiome, however the virome has been neglected due to the lack of appropriate marker genes and analysis benchmarks.
it is imperative that the virome is studied along with the microbiome and the host animal.
the virome, being pathogens of the microbiome, can substantially alter rumen function, which in turn may hinder the ability of the host to utilise feed efficiently.
additionally, as the microbiome in ruminants is much more closely linked to major nutrient acquisition than in humans and mice, a manipulation of the virome  may enable a more substantial manipulation of nutrient acquisition than is possible in monogastric animals.
phages have been suggested as a possible methane mitigation strategy through “bacteriophage therapy”  <cit> .
this strategy requires identification of phage species which target the dominant methane producing archaea in the rumen.
such approaches will benefit from metagenomic virome sequencing by the availability of additional information regarding the dominant phage species in the rumen virome, and will help establish an understanding of the stability or variability of the rumen virome over time, which may affect such therapies.
furthermore, comparison of the virome and microbiome of each rumen sample may shed light on the ecological relationship between phage and bacterium.
for now, this work has illustrated the vast amount of species richness and between animal variation in the rumen virome.
identifying which of these sequences may belong to archaea pathogens is a significant challenge for future work.
studies in other species have found that phages can have a protective role for their host  <cit> , and studies investigating control of bacterial gut populations with phages have been successful  <cit> .
the rumen virome of cattle may be the key to unlocking the secretes of the optimally functioning rumen, and therefore, should be a consideration in the quest to develop the ultimate production animal.
CONCLUSIONS
we have completed the deepest sequencing of the rumen virome to date.
the results indicate large taxonomic diversity between the rumen viromes of australian holstein dairy cattle, and that animals housed together may have more similar viromes than those housed separately.
however, this taxonomic variation is not reflected in the functional characteristics of the rumen virome.
in fact, the rumen virome appears to be functionally conserved between animals.
this suggests that while rumen viral genome sequences diverge rapidly between hosts, functional characteristics are under significant evolutionary constraint.
the observed differences between the rumen virome and the rumen microbiome likely reflect the much narrower set of functions typically encoded in a viral genome compared to a microbial genome.
as mps becomes more widely used to investigate viromes, it will be interesting to observe how these species correlate with changes in the bacterial population, or even if population differences are associated or predictive of key traits such as feed conversion efficiency and methane yield.

 the value of domestic ruminants comes from their ability to convert, by means of rumen fermentation, low-quality forages into high quality, high protein products suitable for human consumption. the rumen, the first section of the ruminant stomach, contains symbiotic microorganisms that breakdown ingested food. these microorganisms include eukarya, bacteria, archaea, and large numbers of virus including bacteriophage. the entire bacteriophage or'phage’ population in a sample is termed a virome. massively parallel sequencing is a technology which allows the generation of several million dna sequences in parallel. mps has been used to investigate the bacterial and archaeal populations of the bovine rumen. however, mps studies on the rumen virome are scant. one study sequenced mbp of viral dna from the rumen of three animals, one lactating, one non lactating and one culled animal. the mps study and earlier studies such as, both suggest that there are large amounts of variation in the bovine rumen virome. however some of the variation may have been due to external sources. since the recent rumen virome study, there have been technological advances that have allowed the volume of sequence data to increase by an order of magnitude. these advances now enable the sequencing of viromes from a greater numbers of animals, resulting in the ability to gain an insight into natural variation between animals. mps virome studies have made it possible to study viral genomes even when the host bacterium cannot be cultured. however, there are significant challenges involving analysis of mps virome data. tools for the analysis of virome samples are becoming more readily available allowing estimates of virome characteristics such as species richness. the combination of published analysis techniques, along with the increased ability to generate data has allowed the field to switch from descriptive analyses to quantitative investigations of how various factors affect the virome. to access the amount of variation in the rumen virome we extracted virus like dna from the rumen fluid of lactating dairy cattle. twelve of the cows were housed together, fed the same diet, had a shared location and were sampled on the same day, allowing us to investigate the magnitude of natural variation in the rumen virome.  assembly and annotation we sequenced virus like particles from the rumen fluid of lactating australian holstein dairy cattle on the hiseq each library contained between and million read pairs. after a combined assembly of all samples, contigs were greater than kbp in length. these contigs were manually inspected for even coverage and annotated. three of the contigs have all the genes necessary for a complete functional phage genome. we mapped our sequence reads back to the complete set of contigs. while up to % of reads mapped to the whole assembly, only % of reads mapped to contigs greater than kbp. this would suggest that although we have assembled up to 57% of the bovine rumen virome, most of what has been assembled is still highly fragmented, and contained in small contigs. t + + + b d *average of all samples from this study. ccannulated animal. tamount of sequence remaining after removal of poor quality bases. +multiple samples from study combined. bbest model as assessed by the catchall program. dbest model that has been corrected using the assumption of overrepresented singletons. five of the largest contigs were observed in all samples, with all observed in the sample from animal. despite over a third of the most common contigs being represented in all samples, the most abundant contig only accounted for % of sequence reads. there was a strong correlation between the sequencing depth of the sample and the percentage of reads that mapped back to the assembly, partly reflecting the fact that the contigs were assembled from the same data. as sequence depth has a strong impact on contig size, it is likely that we have only assembled genomes from the most dominant phage species in the bovine rumen. of the putative genes in the largest contigs, % were assigned an unknown function because all of the significant blast hits were themselves unannotated. genes involved with either dna replication or repair consisted % of putative genes, % of putative genes were structural and % appear to be involved in lysis. completely novel putative genes made up an additional %. these novel and unannotated genes may encode unknown functions including those involved in cell lysis or lytogenic reproduction of the phage. phage may be integrated into the genomes of their host prokraryotes. to look for evidence of this we aligned sequence data from the microbiomes of the same samples from to the largest contigs. all of the largest contigs were observed in one or more microbiome samples. this could be explained by viral contamination of the microbiome. such contamination would result in the same proportions of viral contigs in the microbiome as are observed in the virome. therefore the proportions of reads that map to the viral contigs in the microbiome were compared to the virome. the abundance of two contigs, e and m, were significantly higher in the microbiome than expected under the viral contamination model. this is evidence that these two phage are integrating into the prokaryote genomes. however this hypothesis that cannot be validated until the host genomes are sequenced or the host and phage and isolated and cultured. additionally, contigs a, b, c, e, f, g and n show homology to genes involved in lysis, suggesting that they may be lytic, however this is again speculation until the phage are isolated. variation in contig abundance between animals the twelve co-habited animals’ viromes appear to be dominated by contigs d, e and j, while animal 2202’s virome shows a much more even coverage between contigs. however these contigs only account of < % of the viromes of the co-habited animals. there also appears to be a heterogeneity in the distributions of contigs between animals. contig h has a high abundance in cow, but is almost below detectable limits in the deepest sequenced animal and is undetectable in cow. the evenness observed in the virome of animal possibly hints that the rumen viral community is not dominated by a single species, but has a large number of taxa with a reasonably even distribution. this evenness of distribution was likely not observed in our other samples due to their limited sequence depth resulting in many unassembled species. to assess the distribution on an independent set of contigs we aligned our reads to contigs assembled from the independent set of animals from. between % and % of our reads aligned to the contigs. the distribution of contigs was much more even when these independent contigs were used. a principle component analysis did not separate the independently housed animal from the co-house animal, although as less than 1% of the reads from any virome aligned, this should be interpreted with caution. the evenness of the phage population concurs with the findings of that even the most abundant phage species represent only a small proportion of the rumen virome. our finding that the most common of phage contigs only represent % of the virome is consistent with this. the higher degree of similarity between the viromes of the co-habited animals and animal may suggest that there is transfer of viral species between the hosts within a herd, although was also cannulated, this confounding effect is not possible to resolve without further virome data. still the variation between co-habited animals illustrates that any transfer between animals does not maintain a completely homogeneous herd. whether the within herd variation is related to traits that are related to rumen microbiome functions, such as methane emission levels or bloat susceptibility is yet to be seen, however this dataset shows that there is natural within herd virome variation which should be investigated. future studies may explore the relationship between this variation and host traits such as disease susceptibility and methane emissions. species richness based on our sequence data, it is clear that estimations of rumen phage diversity using electron microscopy of less than species per host summerised in are dramatic underestimations, and the true number of species likely lies closer to the estimate in of between and thousand. to estimate the number of species present in each of our samples, we used catchall. the estimated total number of species were between and million per sample. over estimations of richness can be caused by spurious singletons, and the biological and technical causes for these have been outlined in. to account for spurious singletons we also used a model which corrects for such artefacts. this model reported between and, species per sample. the divergence between these results and those reported in is not surprising given that the authors of catchall consistently report richness higher than phaccs, and claim that phaccs underestimates richness. our assembly contains mbp of assembled sequence, if we assume an average phage genome size of kbp this results in approximately genomes worth of sequence in our assembly. given that we have only assembled half of the sequence in our samples, and the assembled half would be the less diverse than the unassembled half, approximately thousand genomes could be expected as a lower bound for number of species estimations. this rough calculation concurs with the catchall discounted model estimate for cow and together they tend to suggest that previous assessments of rumen phage richness are indeed underestimations. in any case, it is clear that there is a large amount of variation in the species that make up the b. taurus rumen virome. also the large range of estimates, even under the reduced model, suggests that the viromes of some animals are much more species rich than others. this could be an indication of a more diverse prokaryote population in some animals than others. this large range should be considered in future experimental designs, as deeper sequencing of particularly diverse samples will be needed to capture a snapshot of the viral community. comparison to other viromes there are a number of host associated and non host associated viromes sequenced to date. to compare these virome sequence profiles to ours we obtained reads from publically available datasets derived from published studies. here we have only isolated viral dna, therefore we were unable to compare our results to studies such as that by, which isolated rna viruses. we selected four different types of viromes for comparison with our data. the viromes included samples from cattle rumen, human saliva, swine faeces and non host-associated environments. the reads from the published viromes were aligned to our contigs. the other bovine rumen viromes had only a small percentage of reads that mapped to our contigs. when we compared our reads to reads in the virome obtained from the rumen fluid of cattle as previously reported only 2-5% of our reads mapped. this would suggest that there are substantial differences between the viromes observed by and ourselves. these differences could be a result of the phage isolation technique, or possibly the sequencing technology used. it is also possible that the differences were due to real variations, as there was large amounts of variation observed within our samples and it could be expected that cows from other locations and on different diets might be even more distinct. +multiple samples from study combined. *average of all samples from this study. we then compared our reads to the non-bovine viromes. the swine faeces virome, the human saliva virome and aquaculture pond derived virome did not have any reads mapping to our contigs. we then attempted to map our reads to the reads of the above mentioned databases. less than % of our reads mapped to the swine faeces database, while less than % of our reads mapped to the human saliva or pond viromes. there is an observable cline in the amount of our reads that map to these databases as they become less similar to our sample type, from highest from our own assembly, followed by the same sample type, then an alternative species digestive tract, then non gut derived viromes. this cline in reads that align to contigs from different habitats may be a result of the similar prokaryote populations within gut samples. both swine faeces and bovine rumen are dominated by firmicutes and bacteroidetes phyla, while the salivary microbiome is dominated by proteobacteria. hence the viral species in gut samples likely prey mostly on firmicutes and bacteroidetes, which would result in more similarities between the gut derived viromes then between the rumen and non-gut derived viromes. functional characteristics of the rumen virome we also investigated the hypothesis that the functional characteristics of the bovine rumen virome were conserved between animals despite the large amount of sequence variation observed. we assigned out reads to kegg second level pathways. of the assigned second level kegg pathways, nucleotide metabolism and replication and repair combined made up 57% of virome assignments. this is in contrast to the prokaryote metagenomes of rumen fluid from animals from the same location. evenness of second level kegg pathways was significantly lower in the phage sequences compared to the microbe data. this unevenness in the phage dataset is likely due to the lack of genes coding for functions such as carbohydrate metabolism, which the phage relies on the host cell to provide. we then compared the functional characteristics of our sequences to other viromes. a principle component analysis revealed that all viromes clustered, and all microbial metagenomes clustered based on assignments to kegg second level pathways. these analyses reveal how the virome of a sample is functionally distinct from the microbial metagenome, and that the functional characteristics of viromes are more similar to each other than to microbial metagenomes, even when the viromes are taken from different sample types. this is likely a reflection of the limited number of annotatable functions encoded for in the phage metagenome. interestingly the functions coded for in our viromes are not as variable as that observed in human faeces. some of the human faeces variation may be caused by the small number of reads which could be used in the kegg analysis due to the limited percentage of alignments and the starting data volume. n *average of all samples from this study. ccannulated animal. nnumber of second level kegg categories observed in sample’s data. h’ – shannon diversity index. e – evenness. +multiple samples from study combined. the relatively narrow functional characteristics observed in the rumen fluid virome may be in part due to the lack of homology to annotated genes in the databases used for annotation. the potential of novel bacteriophage derived genes to control the bacterial populations of the host will likely be of interest in both a biomedical sense and, in the case of livestock, reducing enteric methane emissions from the rumen. rumen virome importance domestic ruminants are important production animals to people worldwide. their ability to use microbial fermentation to provide energy for food, fibre and power has seen their numbers soar to billion. because of this, researchers have long been interested in the rumen microbiome, however the virome has been neglected due to the lack of appropriate marker genes and analysis benchmarks. it is imperative that the virome is studied along with the microbiome and the host animal. the virome, being pathogens of the microbiome, can substantially alter rumen function, which in turn may hinder the ability of the host to utilise feed efficiently. additionally, as the microbiome in ruminants is much more closely linked to major nutrient acquisition than in humans and mice, a manipulation of the virome may enable a more substantial manipulation of nutrient acquisition than is possible in monogastric animals. phages have been suggested as a possible methane mitigation strategy through “bacteriophage therapy”. this strategy requires identification of phage species which target the dominant methane producing archaea in the rumen. such approaches will benefit from metagenomic virome sequencing by the availability of additional information regarding the dominant phage species in the rumen virome, and will help establish an understanding of the stability or variability of the rumen virome over time, which may affect such therapies. furthermore, comparison of the virome and microbiome of each rumen sample may shed light on the ecological relationship between phage and bacterium. for now, this work has illustrated the vast amount of species richness and between animal variation in the rumen virome. identifying which of these sequences may belong to archaea pathogens is a significant challenge for future work. studies in other species have found that phages can have a protective role for their host, and studies investigating control of bacterial gut populations with phages have been successful. the rumen virome of cattle may be the key to unlocking the secretes of the optimally functioning rumen, and therefore, should be a consideration in the quest to develop the ultimate production animal.  we have completed the deepest sequencing of the rumen virome to date. the results indicate large taxonomic diversity between the rumen viromes of australian holstein dairy cattle, and that animals housed together may have more similar viromes than those housed separately. however, this taxonomic variation is not reflected in the functional characteristics of the rumen virome. in fact, the rumen virome appears to be functionally conserved between animals. this suggests that while rumen viral genome sequences diverge rapidly between hosts, functional characteristics are under significant evolutionary constraint. the observed differences between the rumen virome and the rumen microbiome likely reflect the much narrower set of functions typically encoded in a viral genome compared to a microbial genome. as mps becomes more widely used to investigate viromes, it will be interesting to observe how these species correlate with changes in the bacterial population, or even if population differences are associated or predictive of key traits such as feed conversion efficiency and methane yield. 
many of the putative genes in the assembled contigs showed no homology to previously annotated genes, highlighting the large amount of work still required to fully annotate the functions encoded in viral genomes. we used massively parallel sequencing of virus like particles to investigate the diversity of the rumen virome in thirteen lactating australian holstein dairy cattle all housed in the same location, of which were sampled on the same day. our findings suggest a large amount of between animal variation in the bovine rumen virome and that co-habiting animals may have more similar viromes than non co-habited animals. we report the deepest sequencing to date of the rumen virome. this work highlights the enormous amount of novelty and variation present in the rumen virome. we compared the functional characteristics of our bovine viromes with that of other viromes, as well as rumen microbiomes.
 the bovine rumen hosts a diverse and complex community of eukarya, bacteria, archea and viruses. the rumen viral population has received little attention compared to the rumen microbial population. we used massively parallel sequencing of virus like particles to investigate the diversity of the rumen virome in thirteen lactating australian holstein dairy cattle all housed in the same location, of which were sampled on the same day.  fourteen putative viral sequence fragments over kbp in length were assembled and annotated. many of the putative genes in the assembled contigs showed no homology to previously annotated genes, highlighting the large amount of work still required to fully annotate the functions encoded in viral genomes. the abundance of the contig sequences varied widely between animals, even though the cattle were of the same age, stage of lactation and fed the same diets. additionally the twelve animals which were co-habited shared a number of their dominant viral contigs. we compared the functional characteristics of our bovine viromes with that of other viromes, as well as rumen microbiomes. at the functional level, we found strong similarities between all of the viral samples, which were highly distinct from the rumen microbiome samples.  our findings suggest a large amount of between animal variation in the bovine rumen virome and that co-habiting animals may have more similar viromes than non co-habited animals. we report the deepest sequencing to date of the rumen virome. this work highlights the enormous amount of novelty and variation present in the rumen virome. viromerumenbacteriophagemetagenomics 