BACKGROUND
rna viruses have small, simple genomes, which have a high level of diversity, due to the low-fidelity viral polymerase used for replication.
traditionally, due to their small genomes, ‘genome-walking’ was used to obtain a reference sequence, from which primer pairs can be designed for down-stream use on similar viruses  <cit> .
however, this methodology can take a large amount of time, effort and expense, and be cumbersome, not least because of the variation within virus species, which results in the need to redesign primer pairs and frequent re-optimization of conditions.
in addition to these optimization issues, every novel virus discovered will require ‘genome walking’.
moreover, the introduction of pcr errors using sanger-based sequencing is problematic, particularly when sequencing from cloned dna.
the use of high-fidelity enzymes and sequencing pcr products directly can overcome this issue in part, but errors occurring early in the amplification process will be sequenced incorrectly  <cit> .
these problems can be avoided by utilizing next generation sequencing , a high-throughput sequencing methodology which generates millions of sequences simultaneously from one sample  <cit> .
multiple platforms are available, the two commonly used are roche  <dig> pyrosequencing  and illumina   <cit> .
these platforms were initially developed and applied to mammalian and bacterial genomes, where sanger methods were prohibitively expensive.
because viral genome sequencing is achievable using the traditional pcr/sanger methods, the virology field has had less necessity to embrace this new technology.
the current ngs assays are optimized for large bacterial or mammalian genomes, where nucleic acid is in abundance and obtaining the hundreds of nanograms of nucleic acid required, is relatively simple.
due to the low percentage of viral rna in relation to host rna in any given sample, obtaining the quantity of viral rna required by the protocols can be problematic.
the advantages of ngs technologies in virology are numerous, and its use is becoming more commonplace  <cit> , particularly to detect and characterize pathogens without prior knowledge of their existence, or in association with the sequelae/disease outcome, in primary or cultured material, without the requirement of specific primers  <cit> .
often however, the virus is at such low abundance in these samples that only one or two reads can be obtained, or at best, a number of unlinked contiguous sequences , for which traditional sanger protocols are employed to complete the genome  <cit> .
conversely, known positive samples have been ‘deep sequenced’ using ngs technologies, for a plethora of uses including determining viral heterogeneity, or the effect of the immune system or pharmaceutical drugs on viruses  <cit> .
these deep sequencing methodologies employ the use of viral pcr amplicons to obtain a depth of coverage sufficient to detect variants which occur down to  <dig> % frequency.
between these two approaches, a methodology to obtain full genome sequences directly from known positive clinical specimens or cultured material, without the use of amplicons, viral enrichment or virus concentration is still lacking.
to this end, we describe a broadly applicable approach to obtaining full genome sequences from clinical or cultured samples, which we have successfully applied to lyssavirus infected samples.
lyssaviruses  have negative-sense, single-stranded rna genomes approximately 12 kb long.
traditionally, partial gene sequence  was utilized for viral speciation and phylogenetic analysis.
however, limitations on the information obtained from these partial sequences and a requirement to use full genome sequence as part of the ictv  criteria to propose new lyssavirus species, in combination with recent advances in high-throughput sequencing, have resulted in an increase of full genome sequences available on genbank.
from the submission of the first full length lyssavirus genome sequence of rabv  in  <dig>  <cit>  to the completion of representative genomes from each of the established twelve lyssavirus species  in  <dig>  the commonly used methodology for obtaining the genomes was relatively unchanged, utilizing reverse transcription then either cloning the cdna directly, or cloning amplified pcr products, and sanger sequencing  <cit> .
of the most recently described lyssavirus species, bblv  <cit>  and ikov  <cit> , only the ikov characterization employed ngs technologies to obtain the full genome sequence, pioneering the way forward to obtaining genome sequences without the requirement to design multiple primers necessary for genome walking.
this study describes the optimization of protocols, required for a variety of starting material, describing a robust, simple, reliable methodology to obtain full genome sequences from original clinical material and cultured samples, both from the cell monolayer and the supernatant.
furthermore, we investigate the snps  observed in the sequences obtained.
RESULTS
optimization of the extraction protocol for tissue samples
two different extraction methodologies were compared to investigate the suitability of the rna obtained.
three brain tissue samples  were extracted, using the same amount of starting material, either by trizol® extraction followed by isopropanol precipitation, or rneasy® plus mini kit .
genomic dna depletion was carried out on-column, either as a separate step for the trizol® extracted rna, or as part of the rna extraction procedure, using the rneasy® plus mini kit.
the rna concentration from the rneasy® kit extracted samples were significantly lower than the trizol® extracted equivalents .
the required 200 ng rna , was not achieved for the rneasy® kit samples, or indeed for the rv <dig> trizol® extracted sample.
for these samples the maximum rna  was used with varying success.
* published after sequence was obtained prior to this publication.
† sequence confirmed by sanger sequencing from the bat brain material.
#both brain and tcsn were sequenced.
for analysis see table  <dig>
- data not available.
tcsn tissue culture supernatant; n/a – not applicable as dnase treatment is incorporated in the rna extraction protocol, therefore no concentration before gdna depletion is available; nd - not done as gdna was not depleted from these samples; 1this column contains the rna concentration after rrna depletion for all samples, except rv <dig>  rv <dig> and rv <dig> tcsn where no rrna depletion was undertaken.
†brain either from host, or from one passage in a mouse, *combining these reads resulted in a single genome contig; # combined results from two  <dig> runs of same library; ^ stored in rnalater; a 3’utr not represented; b 5’utr not represented; c 3’ and 5’ utr not represented.
brain tissue samples ordered by concentration after the depletion process.
where rrna depletion was not undertaken , the concentration values in the ribogreen column are directly comparable to the rna extraction concentration.
the number of total reads and viral reads obtained for the rneasy® kit samples were lower in comparison to the trizol® extracted rna, most likely due to the difference in total rna available for these samples.
on the whole, viral rna was not enriched by the rneasy® kit, as the percentage of viral reads was less for rv <dig> and rv <dig>  although for rv <dig> there was a slight increase in viral specific reads.
without exception, none of the rneasy® kit extracted sample reads were sufficient to obtain a single consensus sequence, due to the low number of viral reads obtained.
furthermore, de novo assembly on two of the three samples  failed to align viral reads into contigs for further analysis resulting in only host contigs being identified .
analysis of depletion methodology
regardless of the originating sample  the concentration of the trizol® extracted rna after gdna depletion was significantly less than the original extract rna sample .
the largest reduction was for rv <dig> where rna at  <dig>  ng/μl was depleted to  <dig>  ng/μl  after removal of genomic dna.
interestingly, this sample was part of a cohort of samples that were highly degraded upon receipt, therefore the majority of rna had already been degraded.
otherwise a reduction of concentration between 3-fold and 100-fold was observed .
the subsequent depletion of rrna resulted in a more conservative fold change of concentration between 30-fold  and 2-fold .
we investigated the requirement to deplete gdna and rrna in cultured viral samples after rna extraction, since the amount of cellular material would be minimal in these supernatant preparations.
comparison at the ribogreen stage determined that rv <dig> and rv <dig> depleted samples are 45-fold and 10-fold less than the rv <dig> and rv <dig> non-depleted rna samples respectively.
indeed, for rv <dig> the total amount of rna was too low to obtain 200 ng rna for fragmentation.
the virus titer of rv <dig> and rv <dig> has been calculated previously  <cit>  with rv <dig>  approximately  <dig> log lower than rv <dig>  therefore the difference in the percentage of viral reads is likely to be a reflection of this.
despite the marked difference between the percentage of viral reads of rv <dig> and rv <dig>  the difference within samples regarding whether the rna was depleted or not, is not so obvious.
indeed, for both samples, the rna sequenced directly without depletion provided more viral-specific reads.
the success of the methodology for both tissue material and cell cultured material, particularly the ability to detect viral sequence after de novo assembly, is illustrated in table  <dig>  apart from the column-extracted samples, all sequences obtained were sufficient to obtain viral specific reads either as a single contig, or a number of contigs, which subsequently can be identified by a blast search.
depth of coverage analysis
the depth of viral read coverage of samples from which a single genome contig was obtained was investigated .
brain tissue sample read depths varied from rv <dig>  total viral reads  <dig>  maximum read depth  <dig>  and average read depth of  <dig> ; to rv <dig>  total viral reads  <dig> , maximum read depth  <dig>  and average read depth of  <dig> .
rv <dig> did have a higher number of viral reads, but from two runs, therefore not directly comparable.
read depths from cell cultured viral samples were higher, with average read depths of  <dig> ,  <dig>  and  <dig>  and maximum read depths of  <dig> ,  <dig>  and  <dig>  .
na = not applicable as no snps were observed.
* stop codon.
viral heterogeneity analysis
due to the processes involved, each read is obtained from a single cdna strand, and therefore where multiple reads cover a region viral heterogeneity can be detected within the reads indicating the presence of a heterologous viral population.
although this methodology is not optimized for investigation of low level viral populations, which would require viral read depths of over  <dig> , it is still possible to observe dominant or high level single nucleotide polymorphisms .
even with this dataset, we were able to observe minimum snps  at 1% of the population in cultured material, and 3% in brain tissue samples .
in general, cell cultured samples had more snps than tissue samples, where some tissue samples  had no detectable snps .
of the  <dig> nucleotide substitutions observed,  <dig>  were conservative .
substitutions were identified throughout the genome, although only one was identified outside of a coding region .
at the amino acid level,  <dig>  were synonymous substitutions resulting in no amino acid change .
two stop codon substitutions were identified in this dataset, both in the rv <dig> tissue culture supernatant samples, and both at a low level  equating to a single read .
furthermore, one of these  had two reads which differed from the consensus, one encoding a stop codon, resulting in a truncated nucleoprotein transcript, the other a synonymous change  .
rv <dig>  an arctic-like rabv, had one snp detected in the  <dig> read data at genome position  <dig> .
this snp is located in the glycoprotein gene resulting in an amino acid substitution at position  <dig> ile/thr at a frequency of 18%.
analysis of currently available lyssavirus glycoprotein sequences, indicates this residue is isoleucine in the majority of lyssavirus species.
rv <dig> glycoprotein sequence is available in genbank, deposited separately by two independent laboratories.
no original material is available for analysis and the published sequences are from passaged viruses.
interestingly, alignments of the published sequences and the sequence obtained in this study are 100% identical, apart from this residue, where jq <dig> had ile <dig> and gu <dig> had thr <dig> .
rv <dig>  a us isolate , was genome sequenced using sanger sequencing as part of a larger dataset  <cit> , providing a unique opportunity to analyze the same virus isolate, propagated and sequenced independently.
alignments of the published rv <dig> genome  and the consensus sequence obtained in this study showed 100% identity.
however, the  <dig> data identified a snp at genome position  <dig>  , resulting in a non-synonymous coding change at residue  <dig> resulting in conservative substitution asn/asp <dig> .
the frequency of this variant in the read data was 27% .
rv <dig> was sequenced both from the host brain tissue directly and from cell culture supernatant after  <dig> passages in bhk cells.
only one snp was observed at position  <dig>  a synonymous coding change a/g <dig>  of the  <dig> original brain tissue reads covering this region, 23% were g <dig>  whereas the tissue culture supernatant sample reads  only had g <dig>  to investigate this variation further, specific primers were designed to amplify this region from the original brain sample and the passaged tcsn.
the original brain material pcr amplicon had a read depth of  <dig> reads,  <dig> % g <dig> and  <dig> % a <dig>  the tcsn pcr amplicon had a read depth of  <dig> reads,  <dig> % g <dig> and  <dig> % a <dig>  confirming the consensus sequence data, but indicating the a variant is still present after  <dig> passages, just at a much lower level.
this deep sequence analysis is a useful tool to investigate certain snps of interest indicated from the consensus data.
rv <dig>  a raccoon-dog rabv from estonia, had good coverage across the genome  and also had the most snps observed within a tissue sample .
across the genome,  <dig> snps were detected,  <dig> within the m-gene  and one synonymous change at genome position  <dig>  the remaining two were both silent, one in the glycoprotein gene  and the other in the l-gene  .
although a full genome contig was not obtained for rv <dig>   <dig> contigs covering approximately 90% of the genome were generated.
full genome sequence had previously been generated using overlapping pcr products and sanger sequencing  <cit> .
comparison between the sanger and  <dig> generated sequences revealed 100% match apart from a single nucleotide polymorphism  at genome position  <dig>  which corresponded to substitution gly/glu .
published sequences of all lyssavirus species .
often de novo assembly failed to incorporate reads which contained the genomic termini.
these reads were only incorporated after splicing the missing sequence, from a published similar genomic sequence, to the consensus sequence deduced from the de novo assembly and subsequently mapping reads against the spliced reference using gs mapper.
subsequent to this dataset, in an attempt to increase the population of genomic end viral reads, the  <dig> sample preparation methodology was modified by the addition of 1pmol of n165- <dig> and lracef <dig> at the hexamer cdna synthesis stage, to enhance the cdna population containing the genomic termini.
this modification was trialed on a genome sample highly related to rv <dig>  where the genomic ends were obtained .
this simple modification will be used for future genome sequences.
for samples where the genomic termini were still absent, rt-pcr was performed on the depleted rna, using primers designed against the highly conserved genomic termini to obtain pcr products which were sequenced directly using sanger sequencing.
although this approach resulted in a genome sequence still lacking the sequencing relating to the primer sites, for known lyssavirus species isolates this is acceptable.
however, for novel lyssavirus species prototypes the ends must be deduced either by race  <cit>  or circularization of the rna, followed by rt-pcr  <cit> .
to this end ikov genomic ends were obtained using circularization of the genomic rna  <cit> .
confirming correct sequence and differentiating between indels/homopolymeric repeats
whenever possible, consensus sequences where aligned together with genome sequences from the same lyssavirus species.
any potential insertions or deletions  were investigated by analysis of the flowgrams in the gs program suite.
a limitation of pyrosequencing processing of raw data is runs of homopolymers such as the gaaaaaaa poly a termination signal found at the end of each lyssavirus gene sequence .
figure  <dig> illustrates the discrepancies between individual reads from the same sample in a homopolymeric run, where the signal strength for each base is plotted for each read.
detecting the correct signal in a homopolymer of four or more bases can be problematic, resulting in incorrect base calling.
in general, the more reads covering the genome the more accurate the resulting consensus sequence .
where discrepancies cannot be resolved, or a new virus species is being sequenced, confirmation of the correct sequence using pcr products spanning the region in question sequenced using sanger technologies may be required.
in this dataset, only the proposed new lyssavirus species ikov  required additional pcr confirmation.
each intergenic region length and sequence was confirmed by rt-pcr, with 100% concurrence between the  <dig> consensus sequence and the sanger sequence derived from pcr products .
discussion
our goal was to establish a methodology which enabled sequencing from brain tissue, and cell cultured samples without the use of specific viral enhancement, ultracentrifugation or filtration to identify novel viruses, or obtain genome sequence for known virus species.
it was important to develop a methodology which would not require prior knowledge of the virus being extracted and to use a methodology which is widely used to extract viral rna.
instead of enriching for virus specific rna, we depleted host genomic dna and rrna to increase the percentage of viral specific rna in the sample.
there are a large number of rna extraction methodologies and kits available which can be broadly divided into two categories; 1) phenol/choloform based extraction and 2) column or bead based extraction.
we decided to compare trizol® as a phenol-based system and rneasy® plus mini kit  as a column-based system.
in our hands, the use of spin columns to extract the rna from tissue, removed the majority of rna in process, resulting in less than the 200 ng minimum quantity of total rna recommended for fragmentation and more importantly, too few viral reads were obtained.
the rneasy® plus mini kit  was successfully used as part of the depletion protocol after initial extraction with trizol® with good results.
in this study, less than 200 ng of rna was used for a number of samples, and depending on the sample type less than 200 ng of rna can provide full genome sequence.
trizol® extracted tissue samples rv <dig>  and rv <dig> , not only obtained sufficient reads to cover the genome, but also obtained enough depth of coverage to identify snps .
furthermore, for rv <dig> depleted tissue culture supernatant only 10 ng of rna was fragmented, yet  <dig>  viral specific reads were mapped equating to 41% of the total reads obtained.
these observations are important to illustrate that the methods for library preparation can be further refined for virus sequencing.
the requirement to deplete tissue culture supernatant derived rna was investigated using two 100 ml supernatant samples.
half the rna was subjected to gdna and rrna depletion, whereas the other was not.
the additional preparation time required to deplete the sample was not rewarded with a significant improvement of either proportion of viral-specific reads or read depth, therefore  <dig> sequencing directly from a trizol® extracted rna is the simplest, yet most effective way to obtain viral genome sequence from peg precipitated tissue culture supernatant samples.
however, it is not always possible, or practical to culture viruses in vitro.
furthermore, any passaging of viruses in a cell culture system will apply selective pressures on the virus and, depending on the number of passages necessary to obtain virus at a high enough titer to harvest, will alter the population of virus being sequenced.
indeed, the number of snps observed in the cell cultured rna preparations were greater than the tissue extracted samples .
furthermore, the number of non-conservative substitutions observed  was significantly more in the cultured samples, including the presence of stop codons .
the rv <dig> synonymous snps identified between the brain material and the tcsn passaged  <dig> times in bhk was investigated by designing specific primers to amplify across the snp from both the original and cultured virus samples.
‘deep sequencing’ of this amplicon determined that the dominant viral population changed from g <dig> to a <dig> after  <dig> passages, and although no a <dig> snp reads were observed in the tcsn consensus sequencing they were still present in the population, just at a low level .
although for this example there is no functional relevance for the variation, this approach is useful to determine high proportion snp changes within passaged virus samples using consensus sequencing which can then be targeted directly by deep sequencing the relevant genomic regions, rather than deep sequencing the entire genome.
the genomic termini are often under-represented in the viral reads obtained.
it is likely this is due to a variety of reasons including the process of trimming reads before assembly, the assembly parameters in de novo and mapping programmes as well as biological influences such as mrna over-representing the viral rna population relative to genomic rna and the effect of defective interfering  particles which are often truncated.
the use of primers situated near the genomic termini during the cdna synthesis stages have been shown to improve the number of termini sequences, but have not completely resolved the problem.
sequences generated on the  <dig> platform, due to the way the incorporation of bases are detected by the intensity of light emission, inherently have issues with long homopolymeric repeats.
on the whole, a consensus sequence with  <dig> or more reads, when mapped using an available genome from the same species, can be confidently confirmed by checking the flowgram data.
however, novel viruses, or viruses with a limited number of reads may require additional confirmation by sanger sequencing pcr products, which span the regions in question.
CONCLUSIONS
the application of this methodology to lyssaviruses from brain material and cell cultured samples has been shown to be highly successful.
moreover, we have successfully sequenced the majority of a flavivirus and hantavirus, demonstrating the applicability of the method to other families of viruses .
there is no reason why any cultured virus cannot be peg precipitated, extracted and sequenced using the same methodology.
we have shown for both a high titer virus  and a low titer virus  100 ml of supernatant is more than enough to obtain genome sequence, and to begin to investigate the presence of viral heterogeneity.
further investigations using 5-10 ml of tissue culture supernatant for a number of other cultured lyssaviruses have proved equally as successful .
furthermore, extraction from the cell monolayer  is an excellent source of viral rna.
the only disadvantage is the potential presence of non-functional virus variants presumably present in the cells as partially synthesized viral derivatives.
in this study however, it was virus in the supernatant which appeared to contain defective virion particles, albeit at a low level.
the platform used in this study  has been shown to be reliable and consistent, however, the rna can be prepared for any ngs platform.
indeed we have obtained full genome sequences from a small number of rna samples prepared as described here from brain material on the illumina platform successfully .
the application of ngs sequencing of viruses has begun a new era of virus discovery and characterization of novel viruses and will revolutionize phylogeographic studies in all fields of virology in the coming years.

 rna viruses have small, simple genomes, which have a high level of diversity, due to the low-fidelity viral polymerase used for replication. traditionally, due to their small genomes, ‘genome-walking’ was used to obtain a reference sequence, from which primer pairs can be designed for down-stream use on similar viruses. however, this methodology can take a large amount of time, effort and expense, and be cumbersome, not least because of the variation within virus species, which results in the need to redesign primer pairs and frequent re-optimization of conditions. in addition to these optimization issues, every novel virus discovered will require ‘genome walking’. moreover, the introduction of pcr errors using sanger-based sequencing is problematic, particularly when sequencing from cloned dna. the use of high-fidelity enzymes and sequencing pcr products directly can overcome this issue in part, but errors occurring early in the amplification process will be sequenced incorrectly. these problems can be avoided by utilizing next generation sequencing, a high-throughput sequencing methodology which generates millions of sequences simultaneously from one sample. multiple platforms are available, the two commonly used are roche pyrosequencing and illumina. these platforms were initially developed and applied to mammalian and bacterial genomes, where sanger methods were prohibitively expensive. because viral genome sequencing is achievable using the traditional pcr/sanger methods, the virology field has had less necessity to embrace this new technology. the current ngs assays are optimized for large bacterial or mammalian genomes, where nucleic acid is in abundance and obtaining the hundreds of nanograms of nucleic acid required, is relatively simple. due to the low percentage of viral rna in relation to host rna in any given sample, obtaining the quantity of viral rna required by the protocols can be problematic. the advantages of ngs technologies in virology are numerous, and its use is becoming more commonplace, particularly to detect and characterize pathogens without prior knowledge of their existence, or in association with the sequelae/disease outcome, in primary or cultured material, without the requirement of specific primers. often however, the virus is at such low abundance in these samples that only one or two reads can be obtained, or at best, a number of unlinked contiguous sequences, for which traditional sanger protocols are employed to complete the genome. conversely, known positive samples have been ‘deep sequenced’ using ngs technologies, for a plethora of uses including determining viral heterogeneity, or the effect of the immune system or pharmaceutical drugs on viruses. these deep sequencing methodologies employ the use of viral pcr amplicons to obtain a depth of coverage sufficient to detect variants which occur down to % frequency. between these two approaches, a methodology to obtain full genome sequences directly from known positive clinical specimens or cultured material, without the use of amplicons, viral enrichment or virus concentration is still lacking. to this end, we describe a broadly applicable approach to obtaining full genome sequences from clinical or cultured samples, which we have successfully applied to lyssavirus infected samples. lyssaviruses have negative-sense, single-stranded rna genomes approximately 12 kb long. traditionally, partial gene sequence was utilized for viral speciation and phylogenetic analysis. however, limitations on the information obtained from these partial sequences and a requirement to use full genome sequence as part of the ictv criteria to propose new lyssavirus species, in combination with recent advances in high-throughput sequencing, have resulted in an increase of full genome sequences available on genbank. from the submission of the first full length lyssavirus genome sequence of rabv in to the completion of representative genomes from each of the established twelve lyssavirus species in the commonly used methodology for obtaining the genomes was relatively unchanged, utilizing reverse transcription then either cloning the cdna directly, or cloning amplified pcr products, and sanger sequencing. of the most recently described lyssavirus species, bblv and ikov, only the ikov characterization employed ngs technologies to obtain the full genome sequence, pioneering the way forward to obtaining genome sequences without the requirement to design multiple primers necessary for genome walking. this study describes the optimization of protocols, required for a variety of starting material, describing a robust, simple, reliable methodology to obtain full genome sequences from original clinical material and cultured samples, both from the cell monolayer and the supernatant. furthermore, we investigate the snps observed in the sequences obtained.  optimization of the extraction protocol for tissue samples two different extraction methodologies were compared to investigate the suitability of the rna obtained. three brain tissue samples were extracted, using the same amount of starting material, either by trizol® extraction followed by isopropanol precipitation, or rneasy® plus mini kit. genomic dna depletion was carried out on-column, either as a separate step for the trizol® extracted rna, or as part of the rna extraction procedure, using the rneasy® plus mini kit. the rna concentration from the rneasy® kit extracted samples were significantly lower than the trizol® extracted equivalents. the required 200 ng rna, was not achieved for the rneasy® kit samples, or indeed for the rv trizol® extracted sample. for these samples the maximum rna was used with varying success. * published after sequence was obtained prior to this publication. † sequence confirmed by sanger sequencing from the bat brain material. #both brain and tcsn were sequenced. for analysis see table  - data not available. tcsn tissue culture supernatant; n/a – not applicable as dnase treatment is incorporated in the rna extraction protocol, therefore no concentration before gdna depletion is available; nd - not done as gdna was not depleted from these samples; 1this column contains the rna concentration after rrna depletion for all samples, except rv rv and rv tcsn where no rrna depletion was undertaken. †brain either from host, or from one passage in a mouse, *combining these reads resulted in a single genome contig; # combined results from two runs of same library; ^ stored in rnalater; a 3’utr not represented; b 5’utr not represented; c 3’ and 5’ utr not represented. brain tissue samples ordered by concentration after the depletion process. where rrna depletion was not undertaken, the concentration values in the ribogreen column are directly comparable to the rna extraction concentration. the number of total reads and viral reads obtained for the rneasy® kit samples were lower in comparison to the trizol® extracted rna, most likely due to the difference in total rna available for these samples. on the whole, viral rna was not enriched by the rneasy® kit, as the percentage of viral reads was less for rv and rv although for rv there was a slight increase in viral specific reads. without exception, none of the rneasy® kit extracted sample reads were sufficient to obtain a single consensus sequence, due to the low number of viral reads obtained. furthermore, de novo assembly on two of the three samples failed to align viral reads into contigs for further analysis resulting in only host contigs being identified. analysis of depletion methodology regardless of the originating sample the concentration of the trizol® extracted rna after gdna depletion was significantly less than the original extract rna sample. the largest reduction was for rv where rna at  ng/μl was depleted to  ng/μl after removal of genomic dna. interestingly, this sample was part of a cohort of samples that were highly degraded upon receipt, therefore the majority of rna had already been degraded. otherwise a reduction of concentration between 3-fold and 100-fold was observed. the subsequent depletion of rrna resulted in a more conservative fold change of concentration between 30-fold and 2-fold. we investigated the requirement to deplete gdna and rrna in cultured viral samples after rna extraction, since the amount of cellular material would be minimal in these supernatant preparations. comparison at the ribogreen stage determined that rv and rv depleted samples are 45-fold and 10-fold less than the rv and rv non-depleted rna samples respectively. indeed, for rv the total amount of rna was too low to obtain 200 ng rna for fragmentation. the virus titer of rv and rv has been calculated previously with rv approximately log lower than rv therefore the difference in the percentage of viral reads is likely to be a reflection of this. despite the marked difference between the percentage of viral reads of rv and rv the difference within samples regarding whether the rna was depleted or not, is not so obvious. indeed, for both samples, the rna sequenced directly without depletion provided more viral-specific reads. the success of the methodology for both tissue material and cell cultured material, particularly the ability to detect viral sequence after de novo assembly, is illustrated in table  apart from the column-extracted samples, all sequences obtained were sufficient to obtain viral specific reads either as a single contig, or a number of contigs, which subsequently can be identified by a blast search. depth of coverage analysis the depth of viral read coverage of samples from which a single genome contig was obtained was investigated. brain tissue sample read depths varied from rv total viral reads maximum read depth and average read depth of; to rv total viral reads, maximum read depth and average read depth of. rv did have a higher number of viral reads, but from two runs, therefore not directly comparable. read depths from cell cultured viral samples were higher, with average read depths of, and and maximum read depths of, and. na = not applicable as no snps were observed. * stop codon. viral heterogeneity analysis due to the processes involved, each read is obtained from a single cdna strand, and therefore where multiple reads cover a region viral heterogeneity can be detected within the reads indicating the presence of a heterologous viral population. although this methodology is not optimized for investigation of low level viral populations, which would require viral read depths of over, it is still possible to observe dominant or high level single nucleotide polymorphisms. even with this dataset, we were able to observe minimum snps at 1% of the population in cultured material, and 3% in brain tissue samples. in general, cell cultured samples had more snps than tissue samples, where some tissue samples had no detectable snps. of the nucleotide substitutions observed, were conservative. substitutions were identified throughout the genome, although only one was identified outside of a coding region. at the amino acid level, were synonymous substitutions resulting in no amino acid change. two stop codon substitutions were identified in this dataset, both in the rv tissue culture supernatant samples, and both at a low level equating to a single read. furthermore, one of these had two reads which differed from the consensus, one encoding a stop codon, resulting in a truncated nucleoprotein transcript, the other a synonymous change. rv an arctic-like rabv, had one snp detected in the read data at genome position. this snp is located in the glycoprotein gene resulting in an amino acid substitution at position ile/thr at a frequency of 18%. analysis of currently available lyssavirus glycoprotein sequences, indicates this residue is isoleucine in the majority of lyssavirus species. rv glycoprotein sequence is available in genbank, deposited separately by two independent laboratories. no original material is available for analysis and the published sequences are from passaged viruses. interestingly, alignments of the published sequences and the sequence obtained in this study are 100% identical, apart from this residue, where jq had ile and gu had thr. rv a us isolate, was genome sequenced using sanger sequencing as part of a larger dataset, providing a unique opportunity to analyze the same virus isolate, propagated and sequenced independently. alignments of the published rv genome and the consensus sequence obtained in this study showed 100% identity. however, the data identified a snp at genome position, resulting in a non-synonymous coding change at residue resulting in conservative substitution asn/asp. the frequency of this variant in the read data was 27%. rv was sequenced both from the host brain tissue directly and from cell culture supernatant after passages in bhk cells. only one snp was observed at position a synonymous coding change a/g of the original brain tissue reads covering this region, 23% were g whereas the tissue culture supernatant sample reads only had g to investigate this variation further, specific primers were designed to amplify this region from the original brain sample and the passaged tcsn. the original brain material pcr amplicon had a read depth of reads, % g and % a the tcsn pcr amplicon had a read depth of reads, % g and % a confirming the consensus sequence data, but indicating the a variant is still present after passages, just at a much lower level. this deep sequence analysis is a useful tool to investigate certain snps of interest indicated from the consensus data. rv a raccoon-dog rabv from estonia, had good coverage across the genome and also had the most snps observed within a tissue sample. across the genome, snps were detected, within the m-gene and one synonymous change at genome position the remaining two were both silent, one in the glycoprotein gene and the other in the l-gene. although a full genome contig was not obtained for rv contigs covering approximately 90% of the genome were generated. full genome sequence had previously been generated using overlapping pcr products and sanger sequencing. comparison between the sanger and generated sequences revealed 100% match apart from a single nucleotide polymorphism at genome position which corresponded to substitution gly/glu. published sequences of all lyssavirus species. often de novo assembly failed to incorporate reads which contained the genomic termini. these reads were only incorporated after splicing the missing sequence, from a published similar genomic sequence, to the consensus sequence deduced from the de novo assembly and subsequently mapping reads against the spliced reference using gs mapper. subsequent to this dataset, in an attempt to increase the population of genomic end viral reads, the sample preparation methodology was modified by the addition of 1pmol of n165- and lracef at the hexamer cdna synthesis stage, to enhance the cdna population containing the genomic termini. this modification was trialed on a genome sample highly related to rv where the genomic ends were obtained. this simple modification will be used for future genome sequences. for samples where the genomic termini were still absent, rt-pcr was performed on the depleted rna, using primers designed against the highly conserved genomic termini to obtain pcr products which were sequenced directly using sanger sequencing. although this approach resulted in a genome sequence still lacking the sequencing relating to the primer sites, for known lyssavirus species isolates this is acceptable. however, for novel lyssavirus species prototypes the ends must be deduced either by race or circularization of the rna, followed by rt-pcr. to this end ikov genomic ends were obtained using circularization of the genomic rna. confirming correct sequence and differentiating between indels/homopolymeric repeats whenever possible, consensus sequences where aligned together with genome sequences from the same lyssavirus species. any potential insertions or deletions were investigated by analysis of the flowgrams in the gs program suite. a limitation of pyrosequencing processing of raw data is runs of homopolymers such as the gaaaaaaa poly a termination signal found at the end of each lyssavirus gene sequence. figure  illustrates the discrepancies between individual reads from the same sample in a homopolymeric run, where the signal strength for each base is plotted for each read. detecting the correct signal in a homopolymer of four or more bases can be problematic, resulting in incorrect base calling. in general, the more reads covering the genome the more accurate the resulting consensus sequence. where discrepancies cannot be resolved, or a new virus species is being sequenced, confirmation of the correct sequence using pcr products spanning the region in question sequenced using sanger technologies may be required. in this dataset, only the proposed new lyssavirus species ikov required additional pcr confirmation. each intergenic region length and sequence was confirmed by rt-pcr, with 100% concurrence between the consensus sequence and the sanger sequence derived from pcr products. discussion our goal was to establish a methodology which enabled sequencing from brain tissue, and cell cultured samples without the use of specific viral enhancement, ultracentrifugation or filtration to identify novel viruses, or obtain genome sequence for known virus species. it was important to develop a methodology which would not require prior knowledge of the virus being extracted and to use a methodology which is widely used to extract viral rna. instead of enriching for virus specific rna, we depleted host genomic dna and rrna to increase the percentage of viral specific rna in the sample. there are a large number of rna extraction methodologies and kits available which can be broadly divided into two categories; 1) phenol/choloform based extraction and 2) column or bead based extraction. we decided to compare trizol® as a phenol-based system and rneasy® plus mini kit as a column-based system. in our hands, the use of spin columns to extract the rna from tissue, removed the majority of rna in process, resulting in less than the 200 ng minimum quantity of total rna recommended for fragmentation and more importantly, too few viral reads were obtained. the rneasy® plus mini kit was successfully used as part of the depletion protocol after initial extraction with trizol® with good results. in this study, less than 200 ng of rna was used for a number of samples, and depending on the sample type less than 200 ng of rna can provide full genome sequence. trizol® extracted tissue samples rv and rv, not only obtained sufficient reads to cover the genome, but also obtained enough depth of coverage to identify snps. furthermore, for rv depleted tissue culture supernatant only 10 ng of rna was fragmented, yet viral specific reads were mapped equating to 41% of the total reads obtained. these observations are important to illustrate that the methods for library preparation can be further refined for virus sequencing. the requirement to deplete tissue culture supernatant derived rna was investigated using two 100 ml supernatant samples. half the rna was subjected to gdna and rrna depletion, whereas the other was not. the additional preparation time required to deplete the sample was not rewarded with a significant improvement of either proportion of viral-specific reads or read depth, therefore sequencing directly from a trizol® extracted rna is the simplest, yet most effective way to obtain viral genome sequence from peg precipitated tissue culture supernatant samples. however, it is not always possible, or practical to culture viruses in vitro. furthermore, any passaging of viruses in a cell culture system will apply selective pressures on the virus and, depending on the number of passages necessary to obtain virus at a high enough titer to harvest, will alter the population of virus being sequenced. indeed, the number of snps observed in the cell cultured rna preparations were greater than the tissue extracted samples. furthermore, the number of non-conservative substitutions observed was significantly more in the cultured samples, including the presence of stop codons. the rv synonymous snps identified between the brain material and the tcsn passaged times in bhk was investigated by designing specific primers to amplify across the snp from both the original and cultured virus samples. ‘deep sequencing’ of this amplicon determined that the dominant viral population changed from g to a after passages, and although no a snp reads were observed in the tcsn consensus sequencing they were still present in the population, just at a low level. although for this example there is no functional relevance for the variation, this approach is useful to determine high proportion snp changes within passaged virus samples using consensus sequencing which can then be targeted directly by deep sequencing the relevant genomic regions, rather than deep sequencing the entire genome. the genomic termini are often under-represented in the viral reads obtained. it is likely this is due to a variety of reasons including the process of trimming reads before assembly, the assembly parameters in de novo and mapping programmes as well as biological influences such as mrna over-representing the viral rna population relative to genomic rna and the effect of defective interfering particles which are often truncated. the use of primers situated near the genomic termini during the cdna synthesis stages have been shown to improve the number of termini sequences, but have not completely resolved the problem. sequences generated on the platform, due to the way the incorporation of bases are detected by the intensity of light emission, inherently have issues with long homopolymeric repeats. on the whole, a consensus sequence with or more reads, when mapped using an available genome from the same species, can be confidently confirmed by checking the flowgram data. however, novel viruses, or viruses with a limited number of reads may require additional confirmation by sanger sequencing pcr products, which span the regions in question.  the application of this methodology to lyssaviruses from brain material and cell cultured samples has been shown to be highly successful. moreover, we have successfully sequenced the majority of a flavivirus and hantavirus, demonstrating the applicability of the method to other families of viruses. there is no reason why any cultured virus cannot be peg precipitated, extracted and sequenced using the same methodology. we have shown for both a high titer virus and a low titer virus 100 ml of supernatant is more than enough to obtain genome sequence, and to begin to investigate the presence of viral heterogeneity. further investigations using 5-10 ml of tissue culture supernatant for a number of other cultured lyssaviruses have proved equally as successful. furthermore, extraction from the cell monolayer is an excellent source of viral rna. the only disadvantage is the potential presence of non-functional virus variants presumably present in the cells as partially synthesized viral derivatives. in this study however, it was virus in the supernatant which appeared to contain defective virion particles, albeit at a low level. the platform used in this study has been shown to be reliable and consistent, however, the rna can be prepared for any ngs platform. indeed we have obtained full genome sequences from a small number of rna samples prepared as described here from brain material on the illumina platform successfully. the application of ngs sequencing of viruses has begun a new era of virus discovery and characterization of novel viruses and will revolutionize phylogeographic studies in all fields of virology in the coming years. 
most rna viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of ngs technologies. however, due to the relatively low abundance of viral rna in relation to host rna, rna viruses have proved relatively difficult to sequence using ngs technologies. as representative rna viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare rna from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the roche platform. furthermore, genome sequences were generated from considerably less than 200 ng rna, indicating that manufacturers’ minimum template guidance is conservative. the approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of rna viruses from a variety of sources.
 with the advent of next generation sequencing technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. most rna viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of ngs technologies. however, due to the relatively low abundance of viral rna in relation to host rna, rna viruses have proved relatively difficult to sequence using ngs technologies. here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare rna from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the roche platform.  as representative rna viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. furthermore, genome sequences were generated from considerably less than 200 ng rna, indicating that manufacturers’ minimum template guidance is conservative. in addition to obtaining genome consensus sequence, a high proportion of snps were identified in the majority of samples analyzed.  the approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of rna viruses from a variety of sources. next generation sequencingpyrosequencinglyssavirusgenomernavirus 