BACKGROUND
in the last decade there has been an increasing appreciation that the intestinal microbiota of mammals has a strong influence on host metabolism, physiology and health  <cit> .
metagenomics has enabled large scale studies of these complex microbial communities in the intestine revealing both a qualitative and quantitative picture of the phylogenetic and functional diversity of intestinal microbes  <cit> .
the majority of intestinal metagenomic studies have focused on the bacterial component of the microbiota during states of health and disease.
in recent years viruses, including bacteriophages , from the mammalian intestine have started to receive much attention  <cit> .
the contribution of phages to intestinal microbiota ecology and their potential effects on the mammalian host are just beginning to be elucidated  <cit> .
to unravel the influence of viruses, and particularly bacteriophages, on microbiota ecology and animal host physiology and health, methods allowing quantitative comparison of virus diversity, abundance and function across samples are needed.
sequencing of viromes  is one method that enables such quantitative comparisons  <cit> .
a crucial step for virome sequencing is the purification of vlps from fecal samples.
vlp purification is necessary for the following two reasons.
first, viruses often have very small genomes compared to bacteria and host derived dnas.
therefore, viral dna represents a small percentage of the total dna in a metagenomic sample  <cit> .
this leads to a proportionally low representation of viruses in the obtained sequencing information if complete microbiomes are sequenced.
second, many phages in the intestinal microbiota are integrated into the genomes of their bacterial hosts as dormant lysogenic prophages  <cit> .
by isolating vlps it is possible to distinguish integrated prophage genomes from phage genomes that are associated with viral particles.
while methods for vlp purification from environments such as seawater have been well-analyzed  <cit> , methods for isolating and purifying intestinal viromes are understudied.
in some environments such as the open ocean viral density is low and vlp concentrating methods such as tangential flow filtration or fecl <dig> precipitation have to be used in addition to purification methods to obtain a sufficient density of vlps for sequencing  <cit> .
in intestinal samples, however, viruses are already highly concentrated and thus additional vlp concentration is not necessary  <cit> .
recent studies of intestinal viromes have used several different methods to purify vlps from fecal samples and to prepare the dna for sequencing  <cit> .
while the effects of dna amplification, library preparation and sequencing method on virus metagenomes have been investigated in great detail  <cit> , a critical evaluation of methods for the purification of vlps from intestinal content or feces has, to our knowledge, not yet been conducted.
since phages show great variability in terms of shape, size, buoyant density, resistance to chemical and mechanical stressors, and nucleic acid content  <cit> , it can be expected that the method of purification will strongly influence the degree to which specific phages and other viruses are retained in purified samples.
for example, one method that has been widely used for vlp purification is cesium chloride  density gradient centrifugation, which purifies phages within specific density ranges and discriminates against phages that fall outside of a specified density.
the aim of our study was to evaluate methods for vlp purification from fecal samples which can be applied to samples in a reproducible and quantitative manner.
these methods should be amenable to large sample numbers in parallel to enable the use of replicates.
furthermore, the methods should permit quantitative comparisons of intestinal viromes across multiple individuals and varying treatment groups.
to assess the effects of purification methods on vlp recovery we used an artificial microbiota of known composition.
this artificial microbiota contained six phages and two bacterial species for which complete genome sequences were available.
RESULTS
we used an artificial intestinal microbiota sample consisting of germ-free mouse feces containing six phages  and two bacterial strains .
phages p <dig>  t <dig>  t <dig>  and ɸvpe <dig> represent double-stranded dna  genomes, m <dig> has a linear single-stranded dna  genome, and ɸ <dig> has a segmented double-stranded rna  genome.
the phages were added in equal numbers and the total number of phage particles  equaled the total number of bacteria  added to the sample .
the two bacterial strains were added to the sample at a 1: <dig> ratio relative to each other.
we tested and evaluated four different methods to purify phages from mouse feces for quantitative metagenomic studies .
the four methods, which we designed based on standard protocols used for virus purification  <cit> , included:  removal of microbial cells by filtration + removal of free dna by dnase digestion ,  dithiothreitol treatment to degrade fecal mucus + filtration + dnase ,  filtration + dnase + condensation mediated phage particle precipitation with polyethylene glycol  and  filtration + dnase + cscl density gradient centrifugation to purify phages based on density  .
a fifth treatment group consisted of the total metagenome  of the original, unpurified sample.
since our study focused on the effects of phage purification methods, we used identical dna extraction and library preparation steps for all samples and processed them in parallel.figure  <dig>
schematic diagram of vlp purification methods.
an artificial intestinal microbiota sample was generated by the addition of six phages and two bacterial strains to germ-free mouse feces.
upon homogenization of the mixture, the sample was split into  <dig> equal subsamples.
two subsamples were immediately set aside for total dna isolation .
the remaining  <dig> samples were centrifuged.
upon completion of centrifugation, two samples were treated with  <dig>  mm dithiothreitol .
all eight samples were filtered to remove bacteria and particulates and treated with dnase.
two samples were loaded onto a cscl gradient and phages were banded as described in methods.
the peg precipitation failed due to the formation of a viscous mass that impaired peg removal by buffer exchange.
after collection of the phages from the cscl gradients, all  <dig> samples were extracted for total nucleic acids using phenol/chloroform extraction.
g+, gram-positive; g-, gram-negative.
to test the purification methods we divided the artificial microbiome sample into ten subsamples of equal mass .
eight subsamples were used to carry out the purification methods  in duplicate.
the remaining two subsamples were used for extraction of the total metagenome .
we will use the following abbreviations for the replicate metagenomes throughout the article: fd <dig> and fd <dig> , dtt <dig> and dtt <dig> , cscl <dig> and cscl <dig> , mg <dig> and mg <dig> .
during purification, the peg method failed due to the formation of a viscous high molecular weight compound upon addition of the peg to the sample filtrate.
this precipitate prevented the subsequent removal of peg by buffer exchange and these samples could no longer be processed as desired .
in the future, the peg method could likely be improved by removing peg by chloroform extraction instead of buffer exchange, however, several virus groups are sensitive to chloroform and would thus be lost during peg extraction .
all eight remaining samples were subjected to paired-end sequencing on an illumina hiseq  <dig> sequencer generating ~ <dig> million paired-end reads per sample.
dna recovery
all three working purification methods  yielded <10% of the dna amount extracted from the mg samples .
mg <dig> yielded 636 ng and mg <dig> yielded 459 ng of dna.
the cscl samples had the lowest yield, approximately 20 ng.
yields in the fd samples were around 40 ng, while the dtt samples were intermediate .
most of the reduction in dna between the mg samples and the purified samples is likely due to the removal of bacterial and mouse dna during sample purification.table  <dig>
method overview/summary
fd
dtt
cscl
mg
purification steps
filtration + dnase
dtt + filtration + dnase
filtration + dnase + cscl centrifugation
total metagenome of unpurified sample
sample throughputa
total duration of protocol b
hands on time c
a:number of samples that can be processed by one person in parallel; b:duration of the respective purification protocol from fecal sample to purified vlps, dna extraction time not included; c:hands on time needed for the number of samples that can be processed by one person in parallel ; d:sample number limited by rotor size of ultracentrifuge and number of density gradients that can be set up in parallel in a reasonable amount of time.
purification efficiency, reproducibility and biases of the purification methods based on read coverage
to evaluate the purification methods we mapped the reads from the eight metagenomes against a set of reference sequences consisting of the genomes of the input phages and bacteria plus the genomes of expected contaminants such as mouse, human and the ɸx <dig> phage that is used as an internal control during illumina sequencing .
more than 97% of all reads mapped unambiguously to one of these reference genomes.
the remaining reads either mapped ambiguously to several of the reference genomes or did not map to any of the reference genomes.
the small number of reads that did not map to any of the reference genomes indicates that the content of unknown dna in the samples was small.figure  <dig>
read abundances from artificial intestinal microbiota samples following vlp purification or whole metagenome processing.
relative read abundances   relative read abundances after normalization to genome sizes .
fd, filtration + dnase – replicates 1&2; dtt, dtt + filtration + dnase – replicates 1&2; cscl, filtration + dnase + cscl gradient – replicates 1&2; mg; total metagenome  – replicates 1& <dig>  *denotes organism read abundances with bars too small to be seen in the figure.
in the following we will use differences in relative read abundances between metagenomes as an estimate of differences in relative dna amounts in the sample.
also, for simplicity, we will refer to sequencing reads mapping to the genome of a specific organism or organism group, as < organism > reads e.g.
mouse reads for reads mapping to the mouse genome and phage reads for the reads mapping to the genomes of all added phages .
all three purification methods  led to an average increase of phage reads of more than 20-fold.
while phage reads account for <5% of all reads in the mg samples, they account for >80% of all reads in the purified samples .
the highest percentage of phage reads was achieved in the cscl samples, followed by the dtt samples and then the fd samples.
in terms of purification efficiency the cscl method is the most efficient at removing mouse dna contamination, however, it has other drawbacks that need to be considered .
intra-method reproducibility was observed for the fd and dtt methods, as judged by the relative read abundances of the four phages for which good read coverage was achieved .
the intra-method ratios of relative read abundances for specific phages were between  <dig>  and  <dig> , which is close to the theoretical optimum of  <dig> .
in contrast, much higher variability was observed between the two cscl replicates, where ratios between  <dig>  and  <dig>  were observed .
this higher intra-method variability for the cscl method may have been caused by the fraction collection, which was based on the protocol published by thurber et al.
<cit> .
the reproducibility might be improved by using specialized gradient-harvesting devices for gradient fractionation followed by careful evaluation of fraction densities  <cit> .table  <dig>
intra- and between method variation based on read abundance ratios*
intra-method variation
between method variation
a
fd1/fd2
dtt1/dtt2
cscl1/cscl2
mg1/mg2
fd/dtt
dtt/cscl
cscl/fd
*perfect reproducibility would result in ratios equal to one.
a:read abundance ratios between methods were calculated using the average read abundance in each method.
as expected, variability between methods was higher than intra-method variability.
the ratios of relative read abundances for specific phages between the fd and dtt methods were close to  <dig> indicating reproducible quantitative results between these methods.
in contrast, some of the ratios between the cscl method and the fd and the dtt methods deviated significantly from  <dig> indicating that different purification biases exist between these methods .
in particular, the read abundances of the ɸvpe <dig> and t <dig> phages were greatly reduced in the cscl purified samples .
we also observed that when read abundances were normalized to genome sizes, the ratios between the fd and dtt methods were even closer to  <dig> .
this can be explained by the fact that normalization of read abundance to genome size yields an estimate of relative genome copy number, which is less influenced by small changes in copy number of contaminating dna originating from large genomes .
small changes in copy number of large genomes can influence read abundances significantly, because these genomes yield a read quantity proportional to their size during sequencing.
our results suggest that normalization of read abundance to genome size  can improve between- sample and -method comparability.
although all phages were mixed into the artificial microbiota sample in equal numbers  their representation in the metagenomes differed greatly from an equal distribution.
since input number of phages should roughly correspond to the input genome copy number for each phage, the read abundances normalized to genome size should in theory be equal for all phages.
consequently, the ratios of these read abundances should be close to  <dig> within any given sample.
however, even in the unaltered mg samples, this is not the case .
both the p <dig> and the ɸvpe <dig> phages have much higher read abundances in the mg samples as compared to the t <dig>  t <dig> and m <dig> phages.
for the m <dig> phage this distortion in read abundance can be explained by its ssdna genome .
for the other phages, there are three potential explanations for why the read abundances differ from expected read abundance in the mg samples.
first, phage genome copy number could be misestimated by the pfu counting method.
this method only counts viable phages that are able to infect and lyse their host.
non-viable vlps or free phage dna would not be considered in these measurements.
it is common for phage lysates to contain a large number of non-viable vlps, which contain nucleic acid, but are not able to produce plaques  <cit> .
to check whether the unequal read abundances between the four dsdna phages are caused by a misrepresentation of total vlps by pfu counting we counted total vlps by epifluorescence microscopy and compared them to the pfu counts .
as expected the total vlp count was higher than the pfu count and the vlp/pfu ratios ranged from  <dig>  to  <dig>  for specific phages .
however, based on this data the phage input numbers into the artificial microbiome sample are still close to the 1: <dig> ratio , which we initially determined by pfu counting and thus the large observed differences in read abundances cannot be explained by differences between pfu and vlp counts.
interestingly, the p <dig> phage, which has the highest read abundance in all treatments, was put into the artificial microbiome sample in lowest number based on vlp counting.
the unequal read abundances for phages in the mg samples could, however, be explained by free phage dna, which would not be detected by either vlp or pfu counting.
the fact that the read abundance ratios for the t <dig>  t <dig> and ɸvpe <dig> are close to  <dig> in the fd and dtt samples suggests that the unequal read abundances for these phages in the mg samples were caused by free phage dna that was removed during the purification procedure.
second, the dna extraction method may extract phages with different efficiencies.
however, since the t <dig>  t <dig> and p <dig> phages are structurally similar , this seems an unlikely explanation.
third, phage genomes can carry a variety of dna modifications  <cit>  that could lead to biases during sequencing library preparation.
it was recently shown that some dna modifications can lead to strong exclusion biases during illumina library preparation  <cit> .table  <dig>
purification method biases for specific phages*
fd
dtt
cscl
mg
*in case of absence of purification biases, ratios of size normalized read abundances between phages would be identical in the unpurified control samples  and the purified samples .
differences in phage to phage ratios between the mg samples and the purified samples indicate that different methods discriminate against particular phages .
the most notable difference was a strong reduction of ɸvpe <dig> and t <dig> read abundances by the cscl method.
there are at least three potential explanations for this reduction.
first, the ɸvpe <dig> and t <dig> particles may not have adequately accumulated in the density range extracted from the cscl density gradient.
phage morphology is diverse and phages can vary widely in their buoyant densities, even when structurally related  <cit> .
for example, the p <dig> and t <dig> phages both belong to the family podoviridae, but have been shown to sediment at different density layers in a cscl gradient  <cit> .
therefore, the bias against specific phage may have been introduced by extracting only one fraction from the cscl gradient, however, the extracted density range was rather large, in theory, encompassing the densities of all phages in the sample.
a second explanation for the observed reduction in read abundance in the cscl samples is that some phage types degrade more rapidly in the cscl gradient due to chemical or mechanical stresses.
a third explanation would be that specific phage types rupture due to osmotic shock during buffer exchange releasing their genome.
osmotic shock is a common means to release nucleic acid from viruses  <cit> .
loss of nucleic acid during buffer exchange is unlikely, however, because the  <dig>  mwco ultrafiltration devices used are made to retain nucleic acids >300 bp.
if osmotic rupturing had occurred, the genomic dna of the ɸvpe <dig> and t <dig> bacteriophages  would have remained in the retentate used for nucleic acid extraction.
additionally, it was previously shown for the t <dig> phage that it is resistant to osmotic shock induced rupture  <cit> .
one potential way to alleviate the biases introduced by the cscl method would be to extract a larger density range from the cscl gradient, which may lead to better retrieval of phages, but could also diminish the “cleansing” effect of the gradient by contamination carry over.
overall the biases introduced by the fd and dtt methods are less than in the cscl method.
in conclusion, the high intra-method reproducibility of the fd and dtt methods allows quantitative cross-sample comparisons of phages.
however, caution must be used when drawing conclusions about the abundances of individual phages within samples due to the fact that read abundance estimates within a sample can deviate by more than one order of magnitude from the actual input phage particle number .
removal of bacterial and host dna
all three purification methods were highly successful in removing both bacterial  and mouse genomic dna as compared to the mg samples .
all three purification methods led to a > <dig> -fold enrichment of phage reads to bacterial reads .
the greatest average fold change for phage to bacterial read enrichment was observed in the cscl samples  and the smallest in the fd samples .
these fold changes indicate a removal of > <dig> % of the bacterial dna by each purification method .
host genomic dna is a significant contaminant of fecal material and its presence should be considered when choosing an appropriate vlp purification method.
for our study, the efficiency of removing mouse dna from the samples differed greatly between the three purification methods.
while the fd method only led to a 55-fold change in the ratio of phage reads to mouse reads indicating a removal of  <dig> % of mouse dna, the cscl method led to a 768-fold enrichment indicating a removal of  <dig> % of mouse dna.
the observed discrepancy in removal efficiency of bacterial dna versus mouse dna may be due to a larger fraction of mouse dna existing as free dna in mouse feces , which can easily pass through the filtration membrane, whereas the majority of the bacterial dna is within cells that are efficiently removed by filtration.table  <dig>
removal efficiency of mouse and bacterial dna by different purification methods
fd
dtt
cscl
mg
average % read abundance
phagea
bacteriab
calculations based on average % read abundance
estimated % decrease of read generating mouse dna as compared to mgc
estimated % decrease of read generating bacterial dna as compared to mgc
a:sum of read abundances for all added bacteriophage ; b:sum of read abundances for the two added bacteria – l. monocytogenes and b. thetaiotaomicron; c:calculated based on read abundances normalized to phage read abundance.
an alternative explanation for the higher amount of mouse dna in the purified samples could be the introduction of mouse dna contamination during sample processing.
such processing-contamination has, for example, been shown to occur when using certain types of dna purification columns  <cit> .
however, the observed differences in mouse dna content between purification methods strongly suggest that mouse dna is sample derived.
assuming that free mouse dna is responsible for the presence of mouse reads in the purified metagenomes, this would suggest that free dna was not completely removed by dnase digestion.
there are two potential reasons for this.
it is possible that specific regions of the mouse genome may have been protected from dnase digestion by adhering proteins protecting the dna from degradation or that the conditions chosen for the dnase digestion were not sufficient to achieve a complete removal of mouse dna.
to check whether specific regions of the mouse genome were protected from dnase digestion we mapped the mouse reads to the mouse genome and visualized their location using the integrative genomics viewer software   <cit> .
we found that the reads were evenly distributed along the mouse genome suggesting that incomplete digestion of mouse dna was not due to protection of specific genomic regions.
these data suggest that efforts to remove host dna during the purification of vlps from fecal samples need to be intensified.
this could be achieved by increasing the dnase concentration during digestion.
the dnase concentration that we used in this study was  <dig> u ml− <dig> and corresponds to what is recommended in a standard protocol for phage purification  <cit> ; however, it has been suggested previously that in samples from animal hosts much higher dnase concentrations may be required to remove host dna contamination  <cit> .
other laboratories have used higher dnase concentrations for sequencing analyses of viruses e.g.
<dig> u ml− <dig> for ocean virus metagenomes  <cit>  and  <dig> u ml− <dig> for pcr based analyses of viruses in serum samples  <cit> .
we would thus recommend using higher dnase concentrations for virus purification to achieve more efficient removal of host dna.
additionally, in our study, dnase digestions were performed at room temperature.
digestion can also be done at 37°c and will yield accelerated degradation and greater removal of host dna contamination.
finally, sequencing read data should be “decontaminated” using in silico methods by mapping the reads against a host reference genome to remove sequencing reads of any remaining host dna.
phage genome recovery by genome assembly
to determine if whole phage genomes could be retrieved from the illumina 100 bp paired-end read data, we performed de novo assemblies from all datasets .
we retrieved complete or almost complete genomes for four phages from all datasets , despite the fact that we did not do any assembly procedure optimization .
our assemblies even contained contigs of the ssdna m <dig> phage for which only very few reads were sequenced.
the largest m <dig> contig  was assembled from the fd <dig> sample representing a significant portion of the  <dig>  kbp genome of the m <dig> phage.table  <dig>
assembly statistics
published sequence
fd1
fd2
dtt1
dtt2
cscl1
cscl2
mg1
mg2
p <dig> largest contig a
a:assemblies of the p <dig> genome from our datasets are slightly larger than the reference genome, which is likely due to the quasi-circular nature of the p <dig> genome, which makes it impossible for the assembly algorithm to determine the exact start and end of the genome.
effects of nucleic acid composition of phage genomes
in our study we focused on the recovery of dsdna phages.
nevertheless, we added two phages with non-dsdna genomes  to the artificial microbiota sample to see if these phages could be recovered by any of the tested methods.
to determine the recovery of the ɸ <dig> phage, which has a dsrna genome, we did a cdna synthesis using the fd and mg samples.
the recovered ɸ <dig> rna in these samples was too low and the sequencing library preparation failed.
consequently not a single read was retrieved for ɸ <dig> in any of the purified samples or in the mg samples.
surprisingly, for the m <dig> phage, which has a ssdna genome, a small number of reads were sequenced in all purified samples and the mg samples.
in theory sequencing of ssdna should be prevented by the illumina library preparation protocol which requires dsdna as input  <cit> .
t <dig> dna ligase, commonly used for illumina adapter ligation, works preferentially with dsdna and excludes ssdna.
however, it has been shown that the t <dig> dna ligase can ligate ssdna albeit with a very low efficiency  <cit> , which might explain why small amounts of m <dig> ssdna were sequenced.
this suggests that the tested methods are suitable for qualitatively assessing ssdna viruses, if a large enough number of sequencing reads is generated.
however, to get a clearer picture of rna and ssdna viruses additional steps should be added to these protocols.
several approaches could be used to achieve greater sequencing coverage of ssdna viruses.
first, multiple displacement amplification , which is known to preferentially amplify ssdna virusus, can be used to generate dsdna  <cit> .
mda, however, has the caveat of introducing strong biases and the resulting sequences can only be used for a qualitative assessment of virus diversity and not quantitative analyses  <cit> .
second, random hexamer primers and dna polymerase i can be used to convert ssdna to dsdna  <cit> .
third, ssdna ligase can be used to ligate illumina adapters directly to ssdna during library preparation, however, this method has not been tested on viral dna  <cit> .
for the analysis of rna viruses, rna can be amplified and converted to dsdna by reverse transcriptase  <cit> .
however, care must be taken to avoid rna degradation by nucleases during sample preparation.
furthermore, nucleic acid types can be separated using hydroxyapatite chromatography prior to dsdna generation  <cit> .
CONCLUSIONS
in this study we used artificial microbiota samples, with known bacterial and viral composition, to evaluate four methods for the purification of vlps from feces for quantitative comparisons of intestinal viromes.
one method  failed during the purification procedure.
all other methods succeeded in isolating vlps and are suitable for quantitative sample comparison if one considers their limitations and pitfalls .
there are some notable differences in the ease of use, throughput, and performance of the different methods .
these differences should be considered when choosing the appropriate method.
while the cscl method outperformed the fd and dtt methods in removal efficiency of host derived dna, the fd and dtt methods showed a lower discrimination against specific phage species and also yielded more total dna.
an additional consideration during study design should be the number of samples that will be analyzed.
the fd and dtt methods allow for a much higher sample throughput, because they do not include the time consuming cscl density gradient centrifugation step, which also limits the number of samples that can be processed in parallel .
as discussed above, the lower efficiency of host dna removal in the fd and dtt methods may be alleviated by increasing dnase concentrations during dnase digestion.
in this study we only observed small differences in the performance of the fd and dtt methods.
therefore, the dtt method can be applied to the processing of large scale fecal samples when mucus degradation is required to prevent filter clogging during vlp purification.
a crucial additional consideration for the design of virome studies is the inclusion of appropriate negative controls to be carried from sample purification to metagenome sequencing and in silico analyses  <cit> .

 in the last decade there has been an increasing appreciation that the intestinal microbiota of mammals has a strong influence on host metabolism, physiology and health. metagenomics has enabled large scale studies of these complex microbial communities in the intestine revealing both a qualitative and quantitative picture of the phylogenetic and functional diversity of intestinal microbes. the majority of intestinal metagenomic studies have focused on the bacterial component of the microbiota during states of health and disease. in recent years viruses, including bacteriophages, from the mammalian intestine have started to receive much attention. the contribution of phages to intestinal microbiota ecology and their potential effects on the mammalian host are just beginning to be elucidated. to unravel the influence of viruses, and particularly bacteriophages, on microbiota ecology and animal host physiology and health, methods allowing quantitative comparison of virus diversity, abundance and function across samples are needed. sequencing of viromes is one method that enables such quantitative comparisons. a crucial step for virome sequencing is the purification of vlps from fecal samples. vlp purification is necessary for the following two reasons. first, viruses often have very small genomes compared to bacteria and host derived dnas. therefore, viral dna represents a small percentage of the total dna in a metagenomic sample. this leads to a proportionally low representation of viruses in the obtained sequencing information if complete microbiomes are sequenced. second, many phages in the intestinal microbiota are integrated into the genomes of their bacterial hosts as dormant lysogenic prophages. by isolating vlps it is possible to distinguish integrated prophage genomes from phage genomes that are associated with viral particles. while methods for vlp purification from environments such as seawater have been well-analyzed, methods for isolating and purifying intestinal viromes are understudied. in some environments such as the open ocean viral density is low and vlp concentrating methods such as tangential flow filtration or fecl precipitation have to be used in addition to purification methods to obtain a sufficient density of vlps for sequencing. in intestinal samples, however, viruses are already highly concentrated and thus additional vlp concentration is not necessary. recent studies of intestinal viromes have used several different methods to purify vlps from fecal samples and to prepare the dna for sequencing. while the effects of dna amplification, library preparation and sequencing method on virus metagenomes have been investigated in great detail, a critical evaluation of methods for the purification of vlps from intestinal content or feces has, to our knowledge, not yet been conducted. since phages show great variability in terms of shape, size, buoyant density, resistance to chemical and mechanical stressors, and nucleic acid content, it can be expected that the method of purification will strongly influence the degree to which specific phages and other viruses are retained in purified samples. for example, one method that has been widely used for vlp purification is cesium chloride density gradient centrifugation, which purifies phages within specific density ranges and discriminates against phages that fall outside of a specified density. the aim of our study was to evaluate methods for vlp purification from fecal samples which can be applied to samples in a reproducible and quantitative manner. these methods should be amenable to large sample numbers in parallel to enable the use of replicates. furthermore, the methods should permit quantitative comparisons of intestinal viromes across multiple individuals and varying treatment groups. to assess the effects of purification methods on vlp recovery we used an artificial microbiota of known composition. this artificial microbiota contained six phages and two bacterial species for which complete genome sequences were available.  we used an artificial intestinal microbiota sample consisting of germ-free mouse feces containing six phages and two bacterial strains. phages p t t and ɸvpe represent double-stranded dna genomes, m has a linear single-stranded dna genome, and ɸ has a segmented double-stranded rna genome. the phages were added in equal numbers and the total number of phage particles equaled the total number of bacteria added to the sample. the two bacterial strains were added to the sample at a 1: ratio relative to each other. we tested and evaluated four different methods to purify phages from mouse feces for quantitative metagenomic studies. the four methods, which we designed based on standard protocols used for virus purification, included: removal of microbial cells by filtration + removal of free dna by dnase digestion, dithiothreitol treatment to degrade fecal mucus + filtration + dnase, filtration + dnase + condensation mediated phage particle precipitation with polyethylene glycol and filtration + dnase + cscl density gradient centrifugation to purify phages based on density. a fifth treatment group consisted of the total metagenome of the original, unpurified sample. since our study focused on the effects of phage purification methods, we used identical dna extraction and library preparation steps for all samples and processed them in parallel.figure schematic diagram of vlp purification methods. an artificial intestinal microbiota sample was generated by the addition of six phages and two bacterial strains to germ-free mouse feces. upon homogenization of the mixture, the sample was split into equal subsamples. two subsamples were immediately set aside for total dna isolation. the remaining samples were centrifuged. upon completion of centrifugation, two samples were treated with  mm dithiothreitol. all eight samples were filtered to remove bacteria and particulates and treated with dnase. two samples were loaded onto a cscl gradient and phages were banded as described in methods. the peg precipitation failed due to the formation of a viscous mass that impaired peg removal by buffer exchange. after collection of the phages from the cscl gradients, all samples were extracted for total nucleic acids using phenol/chloroform extraction. g+, gram-positive; g-, gram-negative. to test the purification methods we divided the artificial microbiome sample into ten subsamples of equal mass. eight subsamples were used to carry out the purification methods in duplicate. the remaining two subsamples were used for extraction of the total metagenome. we will use the following abbreviations for the replicate metagenomes throughout the article: fd and fd, dtt and dtt, cscl and cscl, mg and mg. during purification, the peg method failed due to the formation of a viscous high molecular weight compound upon addition of the peg to the sample filtrate. this precipitate prevented the subsequent removal of peg by buffer exchange and these samples could no longer be processed as desired. in the future, the peg method could likely be improved by removing peg by chloroform extraction instead of buffer exchange, however, several virus groups are sensitive to chloroform and would thus be lost during peg extraction. all eight remaining samples were subjected to paired-end sequencing on an illumina hiseq sequencer generating ~ million paired-end reads per sample. dna recovery all three working purification methods yielded <10% of the dna amount extracted from the mg samples. mg yielded 636 ng and mg yielded 459 ng of dna. the cscl samples had the lowest yield, approximately 20 ng. yields in the fd samples were around 40 ng, while the dtt samples were intermediate. most of the reduction in dna between the mg samples and the purified samples is likely due to the removal of bacterial and mouse dna during sample purification.table method overview/summary fd dtt cscl mg purification steps filtration + dnase dtt + filtration + dnase filtration + dnase + cscl centrifugation total metagenome of unpurified sample sample throughputa total duration of protocol b hands on time c a:number of samples that can be processed by one person in parallel; b:duration of the respective purification protocol from fecal sample to purified vlps, dna extraction time not included; c:hands on time needed for the number of samples that can be processed by one person in parallel; d:sample number limited by rotor size of ultracentrifuge and number of density gradients that can be set up in parallel in a reasonable amount of time. purification efficiency, reproducibility and biases of the purification methods based on read coverage to evaluate the purification methods we mapped the reads from the eight metagenomes against a set of reference sequences consisting of the genomes of the input phages and bacteria plus the genomes of expected contaminants such as mouse, human and the ɸx phage that is used as an internal control during illumina sequencing. more than 97% of all reads mapped unambiguously to one of these reference genomes. the remaining reads either mapped ambiguously to several of the reference genomes or did not map to any of the reference genomes. the small number of reads that did not map to any of the reference genomes indicates that the content of unknown dna in the samples was small.figure read abundances from artificial intestinal microbiota samples following vlp purification or whole metagenome processing. relative read abundances relative read abundances after normalization to genome sizes. fd, filtration + dnase – replicates 1&2; dtt, dtt + filtration + dnase – replicates 1&2; cscl, filtration + dnase + cscl gradient – replicates 1&2; mg; total metagenome – replicates 1& *denotes organism read abundances with bars too small to be seen in the figure. in the following we will use differences in relative read abundances between metagenomes as an estimate of differences in relative dna amounts in the sample. also, for simplicity, we will refer to sequencing reads mapping to the genome of a specific organism or organism group, as < organism > reads e.g. mouse reads for reads mapping to the mouse genome and phage reads for the reads mapping to the genomes of all added phages. all three purification methods led to an average increase of phage reads of more than 20-fold. while phage reads account for <5% of all reads in the mg samples, they account for >80% of all reads in the purified samples. the highest percentage of phage reads was achieved in the cscl samples, followed by the dtt samples and then the fd samples. in terms of purification efficiency the cscl method is the most efficient at removing mouse dna contamination, however, it has other drawbacks that need to be considered. intra-method reproducibility was observed for the fd and dtt methods, as judged by the relative read abundances of the four phages for which good read coverage was achieved. the intra-method ratios of relative read abundances for specific phages were between and, which is close to the theoretical optimum of. in contrast, much higher variability was observed between the two cscl replicates, where ratios between and were observed. this higher intra-method variability for the cscl method may have been caused by the fraction collection, which was based on the protocol published by thurber et al.. the reproducibility might be improved by using specialized gradient-harvesting devices for gradient fractionation followed by careful evaluation of fraction densities.table intra- and between method variation based on read abundance ratios* intra-method variation between method variation a fd1/fd2 dtt1/dtt2 cscl1/cscl2 mg1/mg2 fd/dtt dtt/cscl cscl/fd *perfect reproducibility would result in ratios equal to one. a:read abundance ratios between methods were calculated using the average read abundance in each method. as expected, variability between methods was higher than intra-method variability. the ratios of relative read abundances for specific phages between the fd and dtt methods were close to indicating reproducible quantitative results between these methods. in contrast, some of the ratios between the cscl method and the fd and the dtt methods deviated significantly from indicating that different purification biases exist between these methods. in particular, the read abundances of the ɸvpe and t phages were greatly reduced in the cscl purified samples. we also observed that when read abundances were normalized to genome sizes, the ratios between the fd and dtt methods were even closer to. this can be explained by the fact that normalization of read abundance to genome size yields an estimate of relative genome copy number, which is less influenced by small changes in copy number of contaminating dna originating from large genomes. small changes in copy number of large genomes can influence read abundances significantly, because these genomes yield a read quantity proportional to their size during sequencing. our results suggest that normalization of read abundance to genome size can improve between- sample and -method comparability. although all phages were mixed into the artificial microbiota sample in equal numbers their representation in the metagenomes differed greatly from an equal distribution. since input number of phages should roughly correspond to the input genome copy number for each phage, the read abundances normalized to genome size should in theory be equal for all phages. consequently, the ratios of these read abundances should be close to within any given sample. however, even in the unaltered mg samples, this is not the case. both the p and the ɸvpe phages have much higher read abundances in the mg samples as compared to the t t and m phages. for the m phage this distortion in read abundance can be explained by its ssdna genome. for the other phages, there are three potential explanations for why the read abundances differ from expected read abundance in the mg samples. first, phage genome copy number could be misestimated by the pfu counting method. this method only counts viable phages that are able to infect and lyse their host. non-viable vlps or free phage dna would not be considered in these measurements. it is common for phage lysates to contain a large number of non-viable vlps, which contain nucleic acid, but are not able to produce plaques. to check whether the unequal read abundances between the four dsdna phages are caused by a misrepresentation of total vlps by pfu counting we counted total vlps by epifluorescence microscopy and compared them to the pfu counts. as expected the total vlp count was higher than the pfu count and the vlp/pfu ratios ranged from to for specific phages. however, based on this data the phage input numbers into the artificial microbiome sample are still close to the 1: ratio, which we initially determined by pfu counting and thus the large observed differences in read abundances cannot be explained by differences between pfu and vlp counts. interestingly, the p phage, which has the highest read abundance in all treatments, was put into the artificial microbiome sample in lowest number based on vlp counting. the unequal read abundances for phages in the mg samples could, however, be explained by free phage dna, which would not be detected by either vlp or pfu counting. the fact that the read abundance ratios for the t t and ɸvpe are close to in the fd and dtt samples suggests that the unequal read abundances for these phages in the mg samples were caused by free phage dna that was removed during the purification procedure. second, the dna extraction method may extract phages with different efficiencies. however, since the t t and p phages are structurally similar, this seems an unlikely explanation. third, phage genomes can carry a variety of dna modifications that could lead to biases during sequencing library preparation. it was recently shown that some dna modifications can lead to strong exclusion biases during illumina library preparation.table purification method biases for specific phages* fd dtt cscl mg *in case of absence of purification biases, ratios of size normalized read abundances between phages would be identical in the unpurified control samples and the purified samples. differences in phage to phage ratios between the mg samples and the purified samples indicate that different methods discriminate against particular phages. the most notable difference was a strong reduction of ɸvpe and t read abundances by the cscl method. there are at least three potential explanations for this reduction. first, the ɸvpe and t particles may not have adequately accumulated in the density range extracted from the cscl density gradient. phage morphology is diverse and phages can vary widely in their buoyant densities, even when structurally related. for example, the p and t phages both belong to the family podoviridae, but have been shown to sediment at different density layers in a cscl gradient. therefore, the bias against specific phage may have been introduced by extracting only one fraction from the cscl gradient, however, the extracted density range was rather large, in theory, encompassing the densities of all phages in the sample. a second explanation for the observed reduction in read abundance in the cscl samples is that some phage types degrade more rapidly in the cscl gradient due to chemical or mechanical stresses. a third explanation would be that specific phage types rupture due to osmotic shock during buffer exchange releasing their genome. osmotic shock is a common means to release nucleic acid from viruses. loss of nucleic acid during buffer exchange is unlikely, however, because the mwco ultrafiltration devices used are made to retain nucleic acids >300 bp. if osmotic rupturing had occurred, the genomic dna of the ɸvpe and t bacteriophages would have remained in the retentate used for nucleic acid extraction. additionally, it was previously shown for the t phage that it is resistant to osmotic shock induced rupture. one potential way to alleviate the biases introduced by the cscl method would be to extract a larger density range from the cscl gradient, which may lead to better retrieval of phages, but could also diminish the “cleansing” effect of the gradient by contamination carry over. overall the biases introduced by the fd and dtt methods are less than in the cscl method. in conclusion, the high intra-method reproducibility of the fd and dtt methods allows quantitative cross-sample comparisons of phages. however, caution must be used when drawing conclusions about the abundances of individual phages within samples due to the fact that read abundance estimates within a sample can deviate by more than one order of magnitude from the actual input phage particle number. removal of bacterial and host dna all three purification methods were highly successful in removing both bacterial and mouse genomic dna as compared to the mg samples. all three purification methods led to a > -fold enrichment of phage reads to bacterial reads. the greatest average fold change for phage to bacterial read enrichment was observed in the cscl samples and the smallest in the fd samples. these fold changes indicate a removal of > % of the bacterial dna by each purification method. host genomic dna is a significant contaminant of fecal material and its presence should be considered when choosing an appropriate vlp purification method. for our study, the efficiency of removing mouse dna from the samples differed greatly between the three purification methods. while the fd method only led to a 55-fold change in the ratio of phage reads to mouse reads indicating a removal of % of mouse dna, the cscl method led to a 768-fold enrichment indicating a removal of % of mouse dna. the observed discrepancy in removal efficiency of bacterial dna versus mouse dna may be due to a larger fraction of mouse dna existing as free dna in mouse feces, which can easily pass through the filtration membrane, whereas the majority of the bacterial dna is within cells that are efficiently removed by filtration.table removal efficiency of mouse and bacterial dna by different purification methods fd dtt cscl mg average % read abundance phagea bacteriab calculations based on average % read abundance estimated % decrease of read generating mouse dna as compared to mgc estimated % decrease of read generating bacterial dna as compared to mgc a:sum of read abundances for all added bacteriophage; b:sum of read abundances for the two added bacteria – l. monocytogenes and b. thetaiotaomicron; c:calculated based on read abundances normalized to phage read abundance. an alternative explanation for the higher amount of mouse dna in the purified samples could be the introduction of mouse dna contamination during sample processing. such processing-contamination has, for example, been shown to occur when using certain types of dna purification columns. however, the observed differences in mouse dna content between purification methods strongly suggest that mouse dna is sample derived. assuming that free mouse dna is responsible for the presence of mouse reads in the purified metagenomes, this would suggest that free dna was not completely removed by dnase digestion. there are two potential reasons for this. it is possible that specific regions of the mouse genome may have been protected from dnase digestion by adhering proteins protecting the dna from degradation or that the conditions chosen for the dnase digestion were not sufficient to achieve a complete removal of mouse dna. to check whether specific regions of the mouse genome were protected from dnase digestion we mapped the mouse reads to the mouse genome and visualized their location using the integrative genomics viewer software. we found that the reads were evenly distributed along the mouse genome suggesting that incomplete digestion of mouse dna was not due to protection of specific genomic regions. these data suggest that efforts to remove host dna during the purification of vlps from fecal samples need to be intensified. this could be achieved by increasing the dnase concentration during digestion. the dnase concentration that we used in this study was u ml− and corresponds to what is recommended in a standard protocol for phage purification; however, it has been suggested previously that in samples from animal hosts much higher dnase concentrations may be required to remove host dna contamination. other laboratories have used higher dnase concentrations for sequencing analyses of viruses e.g. u ml− for ocean virus metagenomes and u ml− for pcr based analyses of viruses in serum samples. we would thus recommend using higher dnase concentrations for virus purification to achieve more efficient removal of host dna. additionally, in our study, dnase digestions were performed at room temperature. digestion can also be done at 37°c and will yield accelerated degradation and greater removal of host dna contamination. finally, sequencing read data should be “decontaminated” using in silico methods by mapping the reads against a host reference genome to remove sequencing reads of any remaining host dna. phage genome recovery by genome assembly to determine if whole phage genomes could be retrieved from the illumina 100 bp paired-end read data, we performed de novo assemblies from all datasets. we retrieved complete or almost complete genomes for four phages from all datasets, despite the fact that we did not do any assembly procedure optimization. our assemblies even contained contigs of the ssdna m phage for which only very few reads were sequenced. the largest m contig was assembled from the fd sample representing a significant portion of the kbp genome of the m phage.table assembly statistics published sequence fd1 fd2 dtt1 dtt2 cscl1 cscl2 mg1 mg2 p largest contig a a:assemblies of the p genome from our datasets are slightly larger than the reference genome, which is likely due to the quasi-circular nature of the p genome, which makes it impossible for the assembly algorithm to determine the exact start and end of the genome. effects of nucleic acid composition of phage genomes in our study we focused on the recovery of dsdna phages. nevertheless, we added two phages with non-dsdna genomes to the artificial microbiota sample to see if these phages could be recovered by any of the tested methods. to determine the recovery of the ɸ phage, which has a dsrna genome, we did a cdna synthesis using the fd and mg samples. the recovered ɸ rna in these samples was too low and the sequencing library preparation failed. consequently not a single read was retrieved for ɸ in any of the purified samples or in the mg samples. surprisingly, for the m phage, which has a ssdna genome, a small number of reads were sequenced in all purified samples and the mg samples. in theory sequencing of ssdna should be prevented by the illumina library preparation protocol which requires dsdna as input. t dna ligase, commonly used for illumina adapter ligation, works preferentially with dsdna and excludes ssdna. however, it has been shown that the t dna ligase can ligate ssdna albeit with a very low efficiency, which might explain why small amounts of m ssdna were sequenced. this suggests that the tested methods are suitable for qualitatively assessing ssdna viruses, if a large enough number of sequencing reads is generated. however, to get a clearer picture of rna and ssdna viruses additional steps should be added to these protocols. several approaches could be used to achieve greater sequencing coverage of ssdna viruses. first, multiple displacement amplification, which is known to preferentially amplify ssdna virusus, can be used to generate dsdna. mda, however, has the caveat of introducing strong biases and the resulting sequences can only be used for a qualitative assessment of virus diversity and not quantitative analyses. second, random hexamer primers and dna polymerase i can be used to convert ssdna to dsdna. third, ssdna ligase can be used to ligate illumina adapters directly to ssdna during library preparation, however, this method has not been tested on viral dna. for the analysis of rna viruses, rna can be amplified and converted to dsdna by reverse transcriptase. however, care must be taken to avoid rna degradation by nucleases during sample preparation. furthermore, nucleic acid types can be separated using hydroxyapatite chromatography prior to dsdna generation.  in this study we used artificial microbiota samples, with known bacterial and viral composition, to evaluate four methods for the purification of vlps from feces for quantitative comparisons of intestinal viromes. one method failed during the purification procedure. all other methods succeeded in isolating vlps and are suitable for quantitative sample comparison if one considers their limitations and pitfalls. there are some notable differences in the ease of use, throughput, and performance of the different methods. these differences should be considered when choosing the appropriate method. while the cscl method outperformed the fd and dtt methods in removal efficiency of host derived dna, the fd and dtt methods showed a lower discrimination against specific phage species and also yielded more total dna. an additional consideration during study design should be the number of samples that will be analyzed. the fd and dtt methods allow for a much higher sample throughput, because they do not include the time consuming cscl density gradient centrifugation step, which also limits the number of samples that can be processed in parallel. as discussed above, the lower efficiency of host dna removal in the fd and dtt methods may be alleviated by increasing dnase concentrations during dnase digestion. in this study we only observed small differences in the performance of the fd and dtt methods. therefore, the dtt method can be applied to the processing of large scale fecal samples when mucus degradation is required to prevent filter clogging during vlp purification. a crucial additional consideration for the design of virome studies is the inclusion of appropriate negative controls to be carried from sample purification to metagenome sequencing and in silico analyses. 
we compared the following four methods of vlp purification from fecal samples: filtration + dnase, dithiothreitol treatment + filtration + dnase, filtration + dnase + peg precipitation and filtration + dnase + cscl density gradient centrifugation. based on our data we recommend the use of methods or for large scale studies when quantitative comparison of viral abundances across samples is required. in recent years, advances in sequencing technologies and data analysis techniques have enabled detailed metagenomic studies investigating intestinal viromes and their potential contributions to the ecology of the microbiota. viruses are a significant component of the intestinal microbiota in mammals. here we compared and evaluated four methods for vlp purification using artificial intestinal microbiota samples of known bacterial and viral composition. in particular the cscl density gradient centrifugation method, which is frequently used for vlp purification, was most efficient in removing host derived dna, but also showed strong discrimination against specific phages and showed a lower reproducibility of quantitative results. an important component of virome studies is the isolation and purification of virus-like particles from intestinal contents or feces. we observed several differences between methods related to the removal efficiency of bacterial and host dnas and biases against specific phages. a rigorous evaluation of methods for vlp purification is critical as many studies begin to move from descriptive analyses of virus diversity to studies striving to quantitatively compare viral abundances across many samples. several methods have been applied to isolate vlps from intestinal samples, yet to our knowledge, the efficiency and reproducibility between methods have not been explored. therefore, reproducible vlp purification methods which allow for high sample throughput are needed. three of the four tested methods worked well for vlp purification.
 viruses are a significant component of the intestinal microbiota in mammals. in recent years, advances in sequencing technologies and data analysis techniques have enabled detailed metagenomic studies investigating intestinal viromes and their potential contributions to the ecology of the microbiota. an important component of virome studies is the isolation and purification of virus-like particles from intestinal contents or feces. several methods have been applied to isolate vlps from intestinal samples, yet to our knowledge, the efficiency and reproducibility between methods have not been explored. a rigorous evaluation of methods for vlp purification is critical as many studies begin to move from descriptive analyses of virus diversity to studies striving to quantitatively compare viral abundances across many samples. therefore, reproducible vlp purification methods which allow for high sample throughput are needed. here we compared and evaluated four methods for vlp purification using artificial intestinal microbiota samples of known bacterial and viral composition.  we compared the following four methods of vlp purification from fecal samples: filtration + dnase, dithiothreitol treatment + filtration + dnase, filtration + dnase + peg precipitation and filtration + dnase + cscl density gradient centrifugation. three of the four tested methods worked well for vlp purification. we observed several differences between methods related to the removal efficiency of bacterial and host dnas and biases against specific phages. in particular the cscl density gradient centrifugation method, which is frequently used for vlp purification, was most efficient in removing host derived dna, but also showed strong discrimination against specific phages and showed a lower reproducibility of quantitative results.  based on our data we recommend the use of methods or for large scale studies when quantitative comparison of viral abundances across samples is required. the cscl density gradient centrifugation method, while being excellently suited to achieve highly purified samples, in our opinion, should be used with caution when performing quantitative studies. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords virus metagenomicsviral metagenomesvirus-like particlesmicrobiomebacteriophagecscl density gradientissue-copyright-statement© the author 2015 