BACKGROUND
the control of diarrheal diseases remains a constant public health challenge; current estimates predict that diarrhea results in approximately  <dig>  deaths and  <dig>  disability adjusted life years  globally per year  <cit> .
the greatest burden of disease, and consequently the biggest impact on dalys, arises in children under the five years of age residing in countries with a low economic index  <cit> .
diarrhea is a complex syndrome that can be induced by a number of perturbations of the gastrointestinal tract, but the disease is generally associated with specific viruses, bacteria and parasites that induce diarrhea via differing mechanisms.
despite the availability of efficacious vaccines against rotavirus a , this ubiquitous, highly virulent and extremely transmissible virus remains the most common cause of diarrhea in children under the age of two years globally  <cit> .
rov is the suspected etiological agent in 39% and 45% of all hospital admissions related to diarrhea globally and in asia, respectively.
in our setting in vietnam, where vaccine uptake has been limited, rov is estimated to be responsible for between  <dig> and 60% of all childhood diarrheal infections requiring hospitalization  <cit> .
rov is a non-enveloped virus and a member of the reoviridae with a genome comprised of  <dig> segments  of double-stranded rna  of differing lengths  <cit> .
these  <dig> segments encode the six structural  and six non-structural proteins  that constitute a functional, infectious virion.
currently, sequence variation within two of the genes encoding the outer viral capsid proteins  permits a basic differentiation of rov strains  <cit> .
the sequences of the vp <dig>  and the vp <dig>  genes define the g-type and p-type, respectively.
g1p <cit>  is consistently the most frequent rov a g/p type isolated from symptomatic humans globally, but  <dig> other g types and  <dig> alternative p types have been described  <cit> .
g/p typing has historically been considered to be adequate for rov surveillance, epidemiology and for the identification of escapees during vaccine efficacy studies .
however, sequencing of just 2/ <dig> genome segments disregards approximately 81%  of the genome that exists outside these capsid genes, thus limiting our phylogenetic understanding of the other genome segments and the evolutionary processes that may be active across the rov genome.
to address this limitation, a more complex rov classification system using all eleven genomic segments has been established, resulting in a whole genome nomenclature of gx-p-ix-rx-cx-mx-ax-nx-tx-ex-hx, representing vp7-vp4-vp6-vp1-vp2-vp3-nsp1-nsp2-nsp3-nsp4-nsp <dig>  respectively  <cit> .
currently, the purification of pure rov rna suitable for direct genome sequencing is largely dependent on viral culture.
this method is laborious, unreliable and induces bias for sequencing of high yield, cultivable viral particles.
for rov genotyping, rov rna is extracted directly from fecal samples and pcr amplification is performed on the vp <dig> and vp <dig> regions.
this method is fundamentally unsuitable for genome sequencing via next generation sequencing owing to the high amount of contaminating  nucleic acid in the sample, and produces low yields of rov-specific sequence  upon next generation sequencing.
despite the introduction of a whole genome nomenclature for rov, whole genome sequencing is yet to be universally adopted for routine identification, surveillance and phylogenetic studies.
this is because the whole genome sequencing of rov is far from straightforward and relies on the independent amplification of each segment from a positive fecal sample.
the combination of dsrna, fecal extraction as an amplification template, untypeable viruses and highly polymorphic sequences add to this complexity.
the presence of mixed infection of a single sample with multiple rov, reported in approximately 10% of infections in developed countries  <cit>  and 21–48% in low income countries  <cit> , further complicates rov genotyping and sequencing efforts.
the use of sanger sequencing methods, which generate a single consensus sequence based on nucleotide abundance, does not permit the detection of genetic variation or the presence of multiple viruses within a sample, both of which may bias inference related to rov infection and evolution.
random amplification combined with next generation sequencing  methods can be utilized overcome many of the complications that prevent the generation of adequately characterised rov gene sequences representative of the complete diversity present in a single sample.
however, there are still barriers; feces is a complex starting material, containing inhibitors and a wide diversity of organic material, and it is likely that rov dsrna will be of low yield in comparison to nucleic acid from other more copious viruses, prokaryotes, and eukaryotic cells found in the gastrointestinal tract.
to overcome these issues, we present here a novel method suitable for the generation of whole genome sequences of rov directly from human fecal specimens.
RESULTS
a novel method for the purification of rotavirus nucleic acid from fecal samples
to address the limitations surrounding rov nucleic acid purification, we developed a new method utilizing commercial enzyme immunoassay  plates to capture rov particles suitable for rna extraction and amplification.
this procedure is described in detail in the methods; fig.
<dig> shows a flow diagram outlining the various laboratory steps.
briefly, fecal samples testing positive for rov by reverse transcriptase -pcr were captured using eia plates, exogenous nucleic acids removed, and rna extracted from the captured viral particles.
in order to prepare the purified rov rna for genome sequencing the rna was converted to double-stranded cdna prior to standard library preparation necessary for illumina sequencing.fig.
<dig> the procedure for rotavirus capture and nucleic acid preparation prior to genome sequencing.
flow diagram describing the major protocol steps required for the purification of rov and the amplification of rov specific nucleic acid for next generation genome sequencing.
image of illumina miseq obtained from https://assets.illumina.com/content/dam/illumina-marketing/images/systems/miseqdx/web-graphic-miseq-front-comparison-chart.jpg.
clipart image of pipette obtained from http://www.clker.com/clipart-pipette-with-tip.html.
clipart image of 96-well plate obtained from http://cyberuse.com/96-well-plate-template.html.
clipart image of eppendorf tube obtained from http://www.clker.com/clipart-eppendorf-tube-with-open-cap- <dig> html and edited in adobe illustrator
rotavirus purification for genome sequencing is dependent on viral load
we first performed and validated the rov purification method on a human fecal sample that contained a high yield  of the most common rov genotype in human infections, g1p <cit>  .
we found that purification of viral particles on eia plates and the conversion to cdna substantially reduced the yield of rov rna, producing sequential ct values of  <dig> and  <dig>  at each step, respectively .
however, after random amplification  <cit> , the ct value was restored to  <dig> and subsequent genome sequencing on an illumina miseq machine produced > <dig>  reads, of which >50% could be attributed to rov  with >90% genome coverage .
predictably, after performing several serial dilutions, we found the rov purification method was highly sensitive to viral load, with minimal rov sequences produced at a starting ct value of  <dig> and no rov sequences produced when the initial starting ct value was > <dig>  .table  <dig> concentration of rotavirus required for genome sequence generation
validation of method for the genome sequencing of differing rotavirus genotypes
we next aimed to assess the performance of the rov purification and sequencing method on a range  of samples containing common  and less common rov genotypes, these included more “exotic” genotypes such as g26p <cit>  and previously untypeable rov positive samples .
only samples with a primary ct value for rov of < <dig> were chosen.
random amplification and illumina sequencing of all  <dig> samples, including those containing untypable rov, yielded large amounts of sequences associated with rov.
the number of rov reads ranged from ~ <dig>  to > <dig> , <dig>  and the percentage of reads corresponding with rov in the final sequencing data ranged from  <dig> % to >97% .table  <dig> concentration of rotavirus in samples at each step of the capture and amplification process
optimisation of rotavirus cdna amplification for genome sequencing
we measured the ability of the method to produce sequences that covered all  <dig> segments of the rov genome.
we found that, even when the proportion of reads associated with rov was low for some samples, that the genome coverage  was high, with 20/ <dig> of the rov positive samples producing sequences corresponding with >90% coverage of reference genomes ,  <dig> – <dig> %).
however, we additionally found that the resulting sequences were highly influenced by segment length.
the vp <dig>  and vp <dig>  regions exhibited a median coverage of > <dig> %, whilst the smaller segments, such as nsp <dig>  and nsp <dig> , exhibited a median coverage of only  <dig> %  and  <dig> % , respectively .
these data suggest a limitation of the random amplification procedure due to an amplification bias against the shorter rov genome segments.
the underrepresentation of the nsp genes  and vp <dig> in the final genome sequences for three selected samples , vn- <dig>  and vn- <dig> g3p <cit> )) can be observed in the sequence coverage plots in fig.
<dig> table  <dig> sequence coverage of rotavirus genome segments using viral capture and random amplification
vn-0006a
vn-0172a
vn-0341a
vn-0221a
vn-0181a
vn-0058a
vn-0132a
asamples with low coverage chosen for resequencing using both random and specific primers
fig.
<dig> rotavirus nucleic acid enrichment for whole genome sequencing.
circular plots showing the coverage  of each of the  <dig> rov segments  and the coverage depth of each of the segments after illumina sequencing for three rov-positive samples .
plots show coverage and depth following viral capture and random amplification  and following viral capture and amplification with both random and specific primers
to enhance our ability to amplify entire rov genomes from fecal samples, improve the coverage of the shorter genome segments and reduce contaminating sequences from other species, we aimed to enrich the amplification process prior to genome sequencing.
we used a combination of the random primer fr26rvn and new fr26rv specific primers, exploiting conserved terminal sequences for each segment to generate specific primers for each of  <dig> segments .
we selected seven samples  that generated incomplete sequences for the shorter genome segments using the random amplification primers alone and repeated the amplification step with the new primer combinations.
the addition of the primer cocktail substantially improved the production of complete genome sequences.
specifically, we were able to generate enhanced coverage of all of the short segments, with the median coverage of nsp <dig>  nsp <dig> and nsp <dig> increasing from  <dig> to  <dig> %, from  <dig>  to  <dig> % and  <dig> to 86%, respectively .
the coverage plots in fig.
<dig> show the effect of the modified primer cocktail on whole genome amplification and segment sequencing for three selected samples: vn- <dig>  vn- <dig> and vn- <dig> table  <dig> primers used for rotavirus genome enrichment amplification
fr20rv
modified fr26rvn
fr26rv-vp7-f
gccggagctctgcagatat-non ribosomal hexanucleotides  <cit>
gccggagctctgcagatatcggctttaaaag
fr26rv-vp4-f
fr26rv-vp4-r
fr26rv-vp4-f2
gccggagctctgcagatatcggtcacatcctc
gccggagctctgcagatatctggcttcgccat
fr26rv-vp6-f
fr26rv-vp1-fb
fr26rv-vp2-fc
fr26rv-vp3-fe
fr26rv-nsp1-f
fr26rv-nsp2-f
fr26rv-nsp3-f
fr26rv-nsp3-r
gccggagctctgcagatatcacataacgcccctat
fr26rv-nsp4-f1
fr26rv-nsp4-f2
fr26rv-nsp4-r1
gccggagctctgcagatatcgcdtcaattccaac
gccggagctctgcagatatctaagaccrttcc
fr26rv-nsp5-f1
fr26rv-nsp5-f2
fr26rv-nsp5-r1
gccggagctctgcagatatcggcttttaaagcgct
gccggagctctgcagatatcctatctgaatcatc
novel insights into rotavirus infections and phylogenetics
once the purification and amplification for rov was optimised, we performed analysis on the sequences of the  <dig> rov positive samples that had been generated through the modified sequencing protocol.
all samples had high coverage, suitable for further downstream sequence and phylogenetic analyses.
firstly, we used the conventional loci  to genotype the  <dig> samples into g and p types.
we were able to determine the genotype in all  <dig> samples, with 22/ <dig> corresponding with the original predicted genotype that was determined using conventional genotyping methods and 4/ <dig> samples providing discrepant results.
we found that these four samples were identified to have untypeable rov by initial genotyping , while genome sequencing revealed that these infections were induced by rov genotypes g2p <cit> , g2p <cit> , g2p <cit>  and g1p <cit> , respectively.
we further found that 5/ <dig> samples produced genome sequences that included multiple sequences of differing genotypes for one or more individual segments.
hypothesizing co-infection, we examined the varying segments and the potential genome constellations of the sequences generated via the capture-amplification method .
given the segment coverage and the most likely segment combinations to form genome constellations , these samples were found to be probable co-infections of g1p <cit> /g2p <cit> , g3p <cit> /g2p <cit> , g2p <cit> /g3p <cit> , g9p <cit> /g8p <cit>  and g1p <cit> /g8p <cit> .
in all examples, we could detect a major and minor rov genotype in the sequences ), with segment coverage roughly associating with read depth.
notably, in all of these co-infection samples, we did not generate sequences for all segments, particularly the shorter segments which were again underrepresented.
nsp <dig> was missing in the minor rov genotype in all samples and all non-structural protein segments were missing in the minor variant in sample vn- <dig> fig.
<dig> rotavirus coinfection is characterized by a major and a minor population.
dark grey bars and blue lines indicate the percentage sequence coverage and total number of reads , respectively, for each of the  <dig> rov segments for the majority population present in five samples containing coinfecting rov populations.
light grey bars and red lines indicate the percentage sequence coverage and total number of reads , respectively, for each of the  <dig> rotavirus a segments for the minority rotavirus population present in five samples containing coinfecting rotavirus populations
finally, to examine potentially reassortant g1p <cit>  sequences , we performed segment-specific phylogenetic analyses of g1p <cit>  and g2p <cit>  sequences with standard wa-like or ds-1-like backbone constellations  <cit> , including  <dig> samples sequenced herein.
maximum likelihood phylogenies demonstrated strong support for two different lineages  for each of the segments .
this divergence is highlighted in the vp <dig> phylogeny , where the g <dig> and g <dig> genotypes form two distinct, well-supported clades.
the corresponding visualization of genome constellations shows an association between the g <dig> genotype and the ds-1-like backbone as well as the g <dig> genotype and the wa-like backbone for all but four of the vietnamese sequences.
these sequences  instead show g1p <cit>  associated with a ds-1-like backbone.
comparison of the phylogeny of vp <dig> to those of vp <dig> and vp <dig> captures the phylogenetic incongruity between segment-specific phylogenies .
mixing of reassortant virus segments can be additionally observed in the vp7-vp <dig> tanglegram, while the vp7-vp <dig> tanglegram shows vp <dig> and vp <dig> sequences to be associated by genotype, suggesting a common phylogenetic history.
a comparison between vp <dig> and all other segments is shown in additional file 1: figure s <dig>  while reassortment among rov segments is not uncommon, standard rov genotyping only captures reassortment between the vp <dig> and vp <dig> segments, and suggests that these infections resulted from typical g1p <cit>  rov.fig.
<dig> genome constellations suggest intergenotype reassortment in vietnamese g1p <cit>  and g2p <cit>  rotaviruses.
a maximum likelihood phylogeny of vp <dig> gene sequences is shown with genome constellations mapped at the tips of the tree.
standard wa-like genome segments are indicated by light grey blocks, ds-1-like segments by dark grey blocks.
taxa names in bold indicate vietnamese sequences from this study; taxa names in red indicate likely reassortant vietnamese sequences.
asterisks indicate ≥85% bootstrap support at internal nodes of interest
fig.
<dig> phylogenetic incongruity among rotavirus segments confirming genomic reassortment.
diagram outlines g1p <cit>  vp7-vp <dig> segments and the ds-1-like backbone segments in four vietnamese g1p <cit>  rotaviruses.
a tanglegram showing corresponding vp <dig> and vp <dig> segments of g1p <cit>  and g2p <cit>  genotypes.
b tanglegram showing phylogenetic incongruity between the vp <dig> and vp <dig> segments of the g1p <cit>  and g2p <cit>  genotype for four reassortant rotaviruses from vietnam.
sequences from nonreassortant vietnamese rotaviruses are indicated by connection with black lines.
sequences from reassortant vietnamese viruses are indicated by connection with red lines.
segments between which no reassortment is detected  are depicted with reassortant viruses connected by red dashed lines.
asterisks indicate ≥85% bootstrap support at internal nodes of interest
discussion
realising the need for a reliable approach for producing rov genome sequences without the requirement for viral culture, we have developed a methodology that reproducibly generates rov genome sequences directly from infected human fecal specimens, producing nearly complete genomes  for 100% of the  <dig> samples tested.
this represents a significant improvement over previous full genome sequencing approaches utilized for large-scale evolutionary analysis, which yielded nearly complete genomes in less than 45% of sequencing attempts  <cit> .
our novel approach bypasses many of the current restrictions for generating virus-specific genome sequences directly from fecal material.
further, our method reduces contamination with non-rov sequences that may overwhelm the final sequence output, which can occur using non-selective nucleic acid purification and amplification procedures prior to sequencing.
by using diagnostic prospect rov eia plates, with an adapted methodology, we show that rov can be reliably purified in appropriate concentrations for downstream amplification and genomic analyses.
these eia plates are commercially available, and the world health organization  rov surveillance network has included these kits in the who-gsm  catalogue for easy procurement for participating rov surveillance network laboratories.
therefore, our technique could theoretically be rolled out throughout participating rov surveillance network laboratories following routine diagnosis.
we additionally suggest that the presented method is more simplistic than other genome sequencing approaches and could be performed for rov or other viral diarrheal pathogens , in a basic molecular virology/microbiology laboratory with access to a thermal cycling machine.
furthermore, given the high affinity for rov particles on the eia plates and the likely stability post-purification, we predict that rov capture prior to extraction and amplification could be performed at field sites and shipped to a central local or international reference laboratory for direct amplification and genome sequencing.
current rov surveillance is largely performed using g/p typing alone, which is dependent on pcr amplification and sequencing of the genes encoding the vp <dig> and vp <dig> surface antigens, respectively.
g/p typing conventionally utilises sanger sequencing and is unable to determine a g/p type for all rov positive fecal specimens.
indeed, in  <dig>  the who rov surveillance network reported  <dig> % to  <dig> % prevalence of untypable rov strains in fecal specimens depending on the location  <cit> .
our findings suggest that untypable rov strains may be an artefact of methodology created via an inability to generate reliable pcr amplicons for the vp <dig> and vp <dig> genes from fecal extractions.
here we found that 4/ <dig> previously untypable rov strains were actually conventional g2p <cit>  and g1p <cit>  strains for which we could not produce a pcr amplicon suitable for conventional sequencing.
we predict that the purification and genome sequencing of additional untypable samples would generate new insights into the global epidemiology and strain diversity of rov.
our ability to generate genome sequences from a wide range of genotypes suggests that this methodology has a good utility and, given the global introduction of rov vaccination, would assist in the development of a robust and expandable system of molecular epidemiology through routine rov surveillance.
given the global relevance of rov and likely impact of rov immunization in coming years, there is a paucity of genomic data for this ubiquitous rna virus in comparison to other rna viruses that have a dramatic impact on global health.
much of our inference of rov strain circulation, epidemiology and evolution has been derived from current inadequate typing methods.
our data, whilst generated from a small sample size, predicts that coinfection and reassortment are common and likely go undetected at a large scale.
we could identify and confirm  four differing rov combinations contributing to mixed infections .
we additionally identified four reassortant viruses  in this restricted cross-section of rov sequences.
there are limited data regarding both mixed infection and reassortment, although both of these biological processes are thought to play an important role in the generation of new variants and may affect pathogenesis and disease phenotype.
current models of rov evolution predict that while vp <dig> and vp <dig> segments reassort frequently, reassortment is less common amongst the segments comprising the genomic backbone, which likely maintain preferred genome constellations determined by protein-protein interactions across different segments  <cit> .
however, the frequency and genomic dynamics of mixed infection, a prerequisite for reassortment, are poorly understood due to a lack of adequate methods to characterize multiple co-infecting viral segments in a single sample.
the method described here could be utilized to fill this knowledge gap by providing full rov genomic data at an epidemiological scale, which would allow for a more accurate characterization of circulating rov, investigation of mixed infection and reassortment dynamics, and analysis of rov genomic diversity and dynamics pre- and post-vaccine introduction.
there are some limitations to our methodology that may necessitate some optimization prior to the universal acceptance of this sequencing strategy.
first, the generation of double-stranded cdna, whilst reliable, is relatively laborious and could be streamlined by using commercially available kits to provide greater utility in field locations.
second, the methodology proved to be reliable only on samples with a ct value of < <dig>  which may limit the study of prolonged infections and fecal carriage, which have been described to have a lower viral load than acute infections  <cit> .
third, we validated the method on a cohort of young children with symptomatic infections in vietnam and the method may need further testing on archived and non-archived samples from differing locations where rov strain circulation may be more variable.
lastly, the use of illumina sequencing makes the outlined method more costly than current genotyping methods.
however, given the wealth of data generated and the decreasing costs of next generation sequencing, we present the proposed method as a first step toward a reproducible system for investigating global rov molecular epidemiology and surveillance of rov population structures pre- and post-vaccine introduction.
CONCLUSIONS
we have developed and evaluated a genome sequencing method for rov from human fecal specimens using commercial available eia plates and a comparatively simple assortment of molecular biology reagents.
the methodology is reliable but requires further validation in a broader context and may provide new biological and epidemiological insights into rov strain diversity and disease phenotype.

 the control of diarrheal diseases remains a constant public health challenge; current estimates predict that diarrhea results in approximately deaths and disability adjusted life years globally per year. the greatest burden of disease, and consequently the biggest impact on dalys, arises in children under the five years of age residing in countries with a low economic index. diarrhea is a complex syndrome that can be induced by a number of perturbations of the gastrointestinal tract, but the disease is generally associated with specific viruses, bacteria and parasites that induce diarrhea via differing mechanisms. despite the availability of efficacious vaccines against rotavirus a, this ubiquitous, highly virulent and extremely transmissible virus remains the most common cause of diarrhea in children under the age of two years globally. rov is the suspected etiological agent in 39% and 45% of all hospital admissions related to diarrhea globally and in asia, respectively. in our setting in vietnam, where vaccine uptake has been limited, rov is estimated to be responsible for between and 60% of all childhood diarrheal infections requiring hospitalization. rov is a non-enveloped virus and a member of the reoviridae with a genome comprised of segments of double-stranded rna of differing lengths. these segments encode the six structural and six non-structural proteins that constitute a functional, infectious virion. currently, sequence variation within two of the genes encoding the outer viral capsid proteins permits a basic differentiation of rov strains. the sequences of the vp and the vp genes define the g-type and p-type, respectively. g1p is consistently the most frequent rov a g/p type isolated from symptomatic humans globally, but other g types and alternative p types have been described. g/p typing has historically been considered to be adequate for rov surveillance, epidemiology and for the identification of escapees during vaccine efficacy studies. however, sequencing of just 2/ genome segments disregards approximately 81% of the genome that exists outside these capsid genes, thus limiting our phylogenetic understanding of the other genome segments and the evolutionary processes that may be active across the rov genome. to address this limitation, a more complex rov classification system using all eleven genomic segments has been established, resulting in a whole genome nomenclature of gx-p-ix-rx-cx-mx-ax-nx-tx-ex-hx, representing vp7-vp4-vp6-vp1-vp2-vp3-nsp1-nsp2-nsp3-nsp4-nsp respectively. currently, the purification of pure rov rna suitable for direct genome sequencing is largely dependent on viral culture. this method is laborious, unreliable and induces bias for sequencing of high yield, cultivable viral particles. for rov genotyping, rov rna is extracted directly from fecal samples and pcr amplification is performed on the vp and vp regions. this method is fundamentally unsuitable for genome sequencing via next generation sequencing owing to the high amount of contaminating nucleic acid in the sample, and produces low yields of rov-specific sequence upon next generation sequencing. despite the introduction of a whole genome nomenclature for rov, whole genome sequencing is yet to be universally adopted for routine identification, surveillance and phylogenetic studies. this is because the whole genome sequencing of rov is far from straightforward and relies on the independent amplification of each segment from a positive fecal sample. the combination of dsrna, fecal extraction as an amplification template, untypeable viruses and highly polymorphic sequences add to this complexity. the presence of mixed infection of a single sample with multiple rov, reported in approximately 10% of infections in developed countries and 21–48% in low income countries, further complicates rov genotyping and sequencing efforts. the use of sanger sequencing methods, which generate a single consensus sequence based on nucleotide abundance, does not permit the detection of genetic variation or the presence of multiple viruses within a sample, both of which may bias inference related to rov infection and evolution. random amplification combined with next generation sequencing methods can be utilized overcome many of the complications that prevent the generation of adequately characterised rov gene sequences representative of the complete diversity present in a single sample. however, there are still barriers; feces is a complex starting material, containing inhibitors and a wide diversity of organic material, and it is likely that rov dsrna will be of low yield in comparison to nucleic acid from other more copious viruses, prokaryotes, and eukaryotic cells found in the gastrointestinal tract. to overcome these issues, we present here a novel method suitable for the generation of whole genome sequences of rov directly from human fecal specimens.  a novel method for the purification of rotavirus nucleic acid from fecal samples to address the limitations surrounding rov nucleic acid purification, we developed a new method utilizing commercial enzyme immunoassay plates to capture rov particles suitable for rna extraction and amplification. this procedure is described in detail in the methods; fig.  shows a flow diagram outlining the various laboratory steps. briefly, fecal samples testing positive for rov by reverse transcriptase -pcr were captured using eia plates, exogenous nucleic acids removed, and rna extracted from the captured viral particles. in order to prepare the purified rov rna for genome sequencing the rna was converted to double-stranded cdna prior to standard library preparation necessary for illumina sequencing.fig. the procedure for rotavirus capture and nucleic acid preparation prior to genome sequencing. flow diagram describing the major protocol steps required for the purification of rov and the amplification of rov specific nucleic acid for next generation genome sequencing. image of illumina miseq obtained from https://assets.illumina.com/content/dam/illumina-marketing/images/systems/miseqdx/web-graphic-miseq-front-comparison-chart.jpg. clipart image of pipette obtained from http://www.clker.com/clipart-pipette-with-tip.html. clipart image of 96-well plate obtained from http://cyberuse.com/96-well-plate-template.html. clipart image of eppendorf tube obtained from http://www.clker.com/clipart-eppendorf-tube-with-open-cap- html and edited in adobe illustrator rotavirus purification for genome sequencing is dependent on viral load we first performed and validated the rov purification method on a human fecal sample that contained a high yield of the most common rov genotype in human infections, g1p. we found that purification of viral particles on eia plates and the conversion to cdna substantially reduced the yield of rov rna, producing sequential ct values of and at each step, respectively. however, after random amplification, the ct value was restored to and subsequent genome sequencing on an illumina miseq machine produced > reads, of which >50% could be attributed to rov with >90% genome coverage. predictably, after performing several serial dilutions, we found the rov purification method was highly sensitive to viral load, with minimal rov sequences produced at a starting ct value of and no rov sequences produced when the initial starting ct value was >.table concentration of rotavirus required for genome sequence generation validation of method for the genome sequencing of differing rotavirus genotypes we next aimed to assess the performance of the rov purification and sequencing method on a range of samples containing common and less common rov genotypes, these included more “exotic” genotypes such as g26p and previously untypeable rov positive samples. only samples with a primary ct value for rov of < were chosen. random amplification and illumina sequencing of all samples, including those containing untypable rov, yielded large amounts of sequences associated with rov. the number of rov reads ranged from ~ to >, and the percentage of reads corresponding with rov in the final sequencing data ranged from % to >97%.table concentration of rotavirus in samples at each step of the capture and amplification process optimisation of rotavirus cdna amplification for genome sequencing we measured the ability of the method to produce sequences that covered all segments of the rov genome. we found that, even when the proportion of reads associated with rov was low for some samples, that the genome coverage was high, with 20/ of the rov positive samples producing sequences corresponding with >90% coverage of reference genomes, – %). however, we additionally found that the resulting sequences were highly influenced by segment length. the vp and vp regions exhibited a median coverage of > %, whilst the smaller segments, such as nsp and nsp, exhibited a median coverage of only % and %, respectively. these data suggest a limitation of the random amplification procedure due to an amplification bias against the shorter rov genome segments. the underrepresentation of the nsp genes and vp in the final genome sequences for three selected samples, vn- and vn- g3p )) can be observed in the sequence coverage plots in fig.  table sequence coverage of rotavirus genome segments using viral capture and random amplification vn-0006a vn-0172a vn-0341a vn-0221a vn-0181a vn-0058a vn-0132a asamples with low coverage chosen for resequencing using both random and specific primers fig. rotavirus nucleic acid enrichment for whole genome sequencing. circular plots showing the coverage of each of the rov segments and the coverage depth of each of the segments after illumina sequencing for three rov-positive samples. plots show coverage and depth following viral capture and random amplification and following viral capture and amplification with both random and specific primers to enhance our ability to amplify entire rov genomes from fecal samples, improve the coverage of the shorter genome segments and reduce contaminating sequences from other species, we aimed to enrich the amplification process prior to genome sequencing. we used a combination of the random primer fr26rvn and new fr26rv specific primers, exploiting conserved terminal sequences for each segment to generate specific primers for each of segments. we selected seven samples that generated incomplete sequences for the shorter genome segments using the random amplification primers alone and repeated the amplification step with the new primer combinations. the addition of the primer cocktail substantially improved the production of complete genome sequences. specifically, we were able to generate enhanced coverage of all of the short segments, with the median coverage of nsp nsp and nsp increasing from to %, from to % and to 86%, respectively. the coverage plots in fig.  show the effect of the modified primer cocktail on whole genome amplification and segment sequencing for three selected samples: vn- vn- and vn- table primers used for rotavirus genome enrichment amplification fr20rv modified fr26rvn fr26rv-vp7-f gccggagctctgcagatat-non ribosomal hexanucleotides gccggagctctgcagatatcggctttaaaag fr26rv-vp4-f fr26rv-vp4-r fr26rv-vp4-f2 gccggagctctgcagatatcggtcacatcctc gccggagctctgcagatatctggcttcgccat fr26rv-vp6-f fr26rv-vp1-fb fr26rv-vp2-fc fr26rv-vp3-fe fr26rv-nsp1-f fr26rv-nsp2-f fr26rv-nsp3-f fr26rv-nsp3-r gccggagctctgcagatatcacataacgcccctat fr26rv-nsp4-f1 fr26rv-nsp4-f2 fr26rv-nsp4-r1 gccggagctctgcagatatcgcdtcaattccaac gccggagctctgcagatatctaagaccrttcc fr26rv-nsp5-f1 fr26rv-nsp5-f2 fr26rv-nsp5-r1 gccggagctctgcagatatcggcttttaaagcgct gccggagctctgcagatatcctatctgaatcatc novel insights into rotavirus infections and phylogenetics once the purification and amplification for rov was optimised, we performed analysis on the sequences of the rov positive samples that had been generated through the modified sequencing protocol. all samples had high coverage, suitable for further downstream sequence and phylogenetic analyses. firstly, we used the conventional loci to genotype the samples into g and p types. we were able to determine the genotype in all samples, with 22/ corresponding with the original predicted genotype that was determined using conventional genotyping methods and 4/ samples providing discrepant results. we found that these four samples were identified to have untypeable rov by initial genotyping, while genome sequencing revealed that these infections were induced by rov genotypes g2p, g2p, g2p and g1p, respectively. we further found that 5/ samples produced genome sequences that included multiple sequences of differing genotypes for one or more individual segments. hypothesizing co-infection, we examined the varying segments and the potential genome constellations of the sequences generated via the capture-amplification method. given the segment coverage and the most likely segment combinations to form genome constellations, these samples were found to be probable co-infections of g1p /g2p, g3p /g2p, g2p /g3p, g9p /g8p and g1p /g8p. in all examples, we could detect a major and minor rov genotype in the sequences ), with segment coverage roughly associating with read depth. notably, in all of these co-infection samples, we did not generate sequences for all segments, particularly the shorter segments which were again underrepresented. nsp was missing in the minor rov genotype in all samples and all non-structural protein segments were missing in the minor variant in sample vn- fig. rotavirus coinfection is characterized by a major and a minor population. dark grey bars and blue lines indicate the percentage sequence coverage and total number of reads, respectively, for each of the rov segments for the majority population present in five samples containing coinfecting rov populations. light grey bars and red lines indicate the percentage sequence coverage and total number of reads, respectively, for each of the rotavirus a segments for the minority rotavirus population present in five samples containing coinfecting rotavirus populations finally, to examine potentially reassortant g1p sequences, we performed segment-specific phylogenetic analyses of g1p and g2p sequences with standard wa-like or ds-1-like backbone constellations, including samples sequenced herein. maximum likelihood phylogenies demonstrated strong support for two different lineages for each of the segments. this divergence is highlighted in the vp phylogeny, where the g and g genotypes form two distinct, well-supported clades. the corresponding visualization of genome constellations shows an association between the g genotype and the ds-1-like backbone as well as the g genotype and the wa-like backbone for all but four of the vietnamese sequences. these sequences instead show g1p associated with a ds-1-like backbone. comparison of the phylogeny of vp to those of vp and vp captures the phylogenetic incongruity between segment-specific phylogenies. mixing of reassortant virus segments can be additionally observed in the vp7-vp tanglegram, while the vp7-vp tanglegram shows vp and vp sequences to be associated by genotype, suggesting a common phylogenetic history. a comparison between vp and all other segments is shown in additional file 1: figure s while reassortment among rov segments is not uncommon, standard rov genotyping only captures reassortment between the vp and vp segments, and suggests that these infections resulted from typical g1p rov.fig. genome constellations suggest intergenotype reassortment in vietnamese g1p and g2p rotaviruses. a maximum likelihood phylogeny of vp gene sequences is shown with genome constellations mapped at the tips of the tree. standard wa-like genome segments are indicated by light grey blocks, ds-1-like segments by dark grey blocks. taxa names in bold indicate vietnamese sequences from this study; taxa names in red indicate likely reassortant vietnamese sequences. asterisks indicate ≥85% bootstrap support at internal nodes of interest fig. phylogenetic incongruity among rotavirus segments confirming genomic reassortment. diagram outlines g1p vp7-vp segments and the ds-1-like backbone segments in four vietnamese g1p rotaviruses. a tanglegram showing corresponding vp and vp segments of g1p and g2p genotypes. b tanglegram showing phylogenetic incongruity between the vp and vp segments of the g1p and g2p genotype for four reassortant rotaviruses from vietnam. sequences from nonreassortant vietnamese rotaviruses are indicated by connection with black lines. sequences from reassortant vietnamese viruses are indicated by connection with red lines. segments between which no reassortment is detected are depicted with reassortant viruses connected by red dashed lines. asterisks indicate ≥85% bootstrap support at internal nodes of interest discussion realising the need for a reliable approach for producing rov genome sequences without the requirement for viral culture, we have developed a methodology that reproducibly generates rov genome sequences directly from infected human fecal specimens, producing nearly complete genomes for 100% of the samples tested. this represents a significant improvement over previous full genome sequencing approaches utilized for large-scale evolutionary analysis, which yielded nearly complete genomes in less than 45% of sequencing attempts. our novel approach bypasses many of the current restrictions for generating virus-specific genome sequences directly from fecal material. further, our method reduces contamination with non-rov sequences that may overwhelm the final sequence output, which can occur using non-selective nucleic acid purification and amplification procedures prior to sequencing. by using diagnostic prospect rov eia plates, with an adapted methodology, we show that rov can be reliably purified in appropriate concentrations for downstream amplification and genomic analyses. these eia plates are commercially available, and the world health organization rov surveillance network has included these kits in the who-gsm catalogue for easy procurement for participating rov surveillance network laboratories. therefore, our technique could theoretically be rolled out throughout participating rov surveillance network laboratories following routine diagnosis. we additionally suggest that the presented method is more simplistic than other genome sequencing approaches and could be performed for rov or other viral diarrheal pathogens, in a basic molecular virology/microbiology laboratory with access to a thermal cycling machine. furthermore, given the high affinity for rov particles on the eia plates and the likely stability post-purification, we predict that rov capture prior to extraction and amplification could be performed at field sites and shipped to a central local or international reference laboratory for direct amplification and genome sequencing. current rov surveillance is largely performed using g/p typing alone, which is dependent on pcr amplification and sequencing of the genes encoding the vp and vp surface antigens, respectively. g/p typing conventionally utilises sanger sequencing and is unable to determine a g/p type for all rov positive fecal specimens. indeed, in the who rov surveillance network reported % to % prevalence of untypable rov strains in fecal specimens depending on the location. our findings suggest that untypable rov strains may be an artefact of methodology created via an inability to generate reliable pcr amplicons for the vp and vp genes from fecal extractions. here we found that 4/ previously untypable rov strains were actually conventional g2p and g1p strains for which we could not produce a pcr amplicon suitable for conventional sequencing. we predict that the purification and genome sequencing of additional untypable samples would generate new insights into the global epidemiology and strain diversity of rov. our ability to generate genome sequences from a wide range of genotypes suggests that this methodology has a good utility and, given the global introduction of rov vaccination, would assist in the development of a robust and expandable system of molecular epidemiology through routine rov surveillance. given the global relevance of rov and likely impact of rov immunization in coming years, there is a paucity of genomic data for this ubiquitous rna virus in comparison to other rna viruses that have a dramatic impact on global health. much of our inference of rov strain circulation, epidemiology and evolution has been derived from current inadequate typing methods. our data, whilst generated from a small sample size, predicts that coinfection and reassortment are common and likely go undetected at a large scale. we could identify and confirm four differing rov combinations contributing to mixed infections. we additionally identified four reassortant viruses in this restricted cross-section of rov sequences. there are limited data regarding both mixed infection and reassortment, although both of these biological processes are thought to play an important role in the generation of new variants and may affect pathogenesis and disease phenotype. current models of rov evolution predict that while vp and vp segments reassort frequently, reassortment is less common amongst the segments comprising the genomic backbone, which likely maintain preferred genome constellations determined by protein-protein interactions across different segments. however, the frequency and genomic dynamics of mixed infection, a prerequisite for reassortment, are poorly understood due to a lack of adequate methods to characterize multiple co-infecting viral segments in a single sample. the method described here could be utilized to fill this knowledge gap by providing full rov genomic data at an epidemiological scale, which would allow for a more accurate characterization of circulating rov, investigation of mixed infection and reassortment dynamics, and analysis of rov genomic diversity and dynamics pre- and post-vaccine introduction. there are some limitations to our methodology that may necessitate some optimization prior to the universal acceptance of this sequencing strategy. first, the generation of double-stranded cdna, whilst reliable, is relatively laborious and could be streamlined by using commercially available kits to provide greater utility in field locations. second, the methodology proved to be reliable only on samples with a ct value of < which may limit the study of prolonged infections and fecal carriage, which have been described to have a lower viral load than acute infections. third, we validated the method on a cohort of young children with symptomatic infections in vietnam and the method may need further testing on archived and non-archived samples from differing locations where rov strain circulation may be more variable. lastly, the use of illumina sequencing makes the outlined method more costly than current genotyping methods. however, given the wealth of data generated and the decreasing costs of next generation sequencing, we present the proposed method as a first step toward a reproducible system for investigating global rov molecular epidemiology and surveillance of rov population structures pre- and post-vaccine introduction.  we have developed and evaluated a genome sequencing method for rov from human fecal specimens using commercial available eia plates and a comparatively simple assortment of molecular biology reagents. the methodology is reliable but requires further validation in a broader context and may provide new biological and epidemiological insights into rov strain diversity and disease phenotype. 
our approach had a 100% success rate, producing >90% genome coverage for diverse rov present in fecal samples. to overcome these limitations, we developed a novel rov capture and genome sequencing method combining commercial enzyme immunoassay plates and a set of routinely used reagents.
 genomic characterization of rotavirus has not been adopted at large-scale due to the complexity of obtaining sequences for all segments, particularly when feces are used as starting material. methods to overcome these limitations, we developed a novel rov capture and genome sequencing method combining commercial enzyme immunoassay plates and a set of routinely used reagents.  our approach had a 100% success rate, producing >90% genome coverage for diverse rov present in fecal samples.  this method provides a novel, reproducible and comparatively simple approach for genomic rov characterization and could be scaled-up for use in global rov surveillance systems. trial registration current controlled trials isrctn date of registration: 14/06/2012 electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rotavirus agenomicsphylogeneticsgenome sequencingreassortmentco-infectionantibody capturethe wellcome trust and the royal society100087/z/12/zbaker stephen oak foundation wellcome trust wt/093724issue-copyright-statement© the author 2017 