BACKGROUND
hepatitis c virus  is a significant human pathogen affecting nearly 3 % of the world’s population, and is a leading cause of chronic liver diseases including cirrhosis and hepatocellular carcinoma  <cit> .
hcv is a member of the flaviviridae family and has a single stranded rna genome that is  <dig>  kb in length with positive polarity.
the genome contains a single open-reading frame and encodes a precursor polyprotein of approximately  <dig> amino acid residues.
within an infected cell, this polyprotein is processed by cellular and host proteases to yield ten structural  and non-structural  proteins  <cit> .
the viral rna-dependent rna polymerase, or ns5b, is a key enzyme in the hcv replication complex within an infected cell, and is responsible for the production of nascent genomes for packaging into new virions.
the polymerase is highly error-prone  <cit> , a feature common to many rna viruses, and as a result hcv exists as seven distinct genotypes , which differ by up to 35 % at the nucleotide level  <cit> .
within each genotype, viruses have been further classified into subtypes  with about 20 % inter-subtype nucleotide divergence  <cit> .
the genotypes are unevenly distributed across the globe, with genotypes  <dig> and  <dig> being the most prevalent  <cit> , and genotype  <dig> being the rarest having only been detected once in canada from a central african immigrant  <cit> .
with the advent of direct-acting antivirals  for the treatment of hcv infections, there is a need to monitor the emergence of resistance-associated variants before and after treatment.
while testing specific regions of the hcv genome using both consensus and next generation sequencing  has enabled such monitoring in daa therapies, including agents targeting ns <dig>  ns5a and ns5b  <cit> , the emerging daa combination regimens emphasise the necessity to simultaneously screen multiple genes of the viral genome in a simple, cost-effective manner.
furthermore, there is a need to detect whether individual viral variants carry multiple polymorphisms conferring resistance against all daas in a combination regimen, therefore increasing the chances of viral persistence against such therapies.
in the past, such analysis was possible only by cloning and consensus sequencing.
however, as the ngs technologies continue to improve read length  the range for covariant studies continues to increase.
in addition to monitoring viral variants associated with drug resistance, methods for the molecular amplification and sequencing of hcv rna have also been instrumental in characterising hcv infections, including studies seeking to understand virus transmission and within-host evolution .
previously, these methods focused on specific regions of the hcv genome, or analysed the entire genome in separate fragments, which is laborious, cost-prohibitive and leads to analysis issues, including uneven coverage due to amplicon pooling, artificial recombinants during genome assemby and multiple pcr primer bias.
therefore, there is a need for sensitive methods that can amplify the entire viral genome from all gts in clinical samples.
a few reports have described the amplification of near full-length hcv genomes, however these were either limited by the sensitivity of the methodology or coverage of all major genotypes .
recently, rna sequencing technology has been applied to sequence hcv in a non-specific manner  <cit> .
while these methods offer the advantage of reduced primer bias and reduced upstream labour, they require increased labour in data analysis, have reduced sensitivity and increased overall cost per sample  <cit> .
here, we describe a simple method which allows sensitive amplification of near full-length hcv genomes from gt  <dig> to  <dig>  using this method, 90 %  of a set of hcv-infected clinical samples were successfully amplified and sequenced using an ngs approach.
as a proof of principle, the method was also applied to amplify and sequence near full-length hcv genomes from two subjects co-infected with multiple genotypes.
the ability to generate near full-length quasispecies sequence was tested with one amplicon using the pacbio rs ii platform.
methods
cohort
stored plasma samples positive for hcv gt <dig>   <dig>   <dig> and  <dig> were made available from the hepatitis c incidence and transmission study in prisons and community cohort , which is a prospective cohort of hcv seronegative and hcv rna negative subjects in new south wales, australia  <cit> .
stored plasma samples positive for hcv gt  <dig> and  <dig> were from academisch medisch centrum  patients identified with hcv infection.
in all subjects, hcv infection was confirmed by detection of hcv-specific antibody and rna.
in the hits cohort, hcv antibody testing was performed using the qualitative abbott architect anti-hcv chemiluminescent microparticle immunoassay .
for the amc patients hcv antibody testing was performed using the axsym hcv  <dig>  serology test .
for all subjects quantitative hcv rna detection was performed with the cobas ampliprep/cobas taqman hcv assay .
ethics statement
for the hits samples ethical approvals were obtained from human research ethics committees of justice health , new south wales department of corrective services , and the university of new south wales , all located in sydney, australia.
written informed consent was obtained from the participants.
for the dutch samples, the study was performed according to the dutch federa code of conduct for responsible use of human tissue and medical research  <dig>
genotyping and detection of multiple infection
genotype determination and detection of multiple hcv genotype infection was performed on a region of the core as previously described  <cit> .
primer design
full-length genome sequences representing each of the six hcv genotypes were downloaded from genbank and used for primer design.
this included  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> different strains for gts 1– <dig> respectively.
to choose primer binding sites we manually scanned the 3′ end of the genome for regions >20 bp with a minimum of 90 % identity.
to improve sequence identity of the primer with the viral variants, degenerates bases were inserted where needed.
to improve binding efficiency degenerate bases were not added to the last  <dig> nucleotides at the 3′ end of the primer and where possible primers had c’s or g’s at the 3′ base.
jfh- <dig> rna t <dig> transcripts
t <dig> rna transcripts for full-length cell culture derived hcv genotype 2a variant, jfh- <dig>  were generated as previously described  <cit> .
rna concentration by ultracentrifugation
for concentration of hcv from plasma samples,  <dig> –1 ml of plasma was thawed and centrifuged at  <dig>  g for 10 min at 4 °c.
supernatants were transferred and centrifuged at  <dig>  g for  <dig>  h at 4 °c after which the pellet was re-suspended in 140 μl of 1× pbs.
viral rna was then extracted from the sample as outlined below.
rna extraction
viral rna was extracted from 140 μl of plasma using the qiamp viral rna kit according to manufacturers’ instructions , with the following modifications: ambion® linear acrylamide  was used instead of the carrier rna provided in the kit; sample lysis was performed by inverting tubes instead of vortexing; and the speed of centrifugation was reduced to  <dig>  g for all steps except the final wash which was carried out at  <dig>  g. finally, rna was eluted in 50 μl of rna storage solution  and stored at −80 °c.
reverse transcription
near full-length hcv cdna was synthesized from viral rna using the superscript iii  first-strand synthesis system  and a pan-genotype primer .
before commencing reverse-transcription , 7 μl of rna, 1 μl of 10 μm primer and 1 μl of 10 mm dntps  were mixed and incubated in a thermocycler  at 65 °c for  <dig> mins and then placed immediately on ice for 1 min.
rt was then initiated with the addition of rt reaction mix to a 20 μl final volume at a final concentration 1x rt buffer, 5 mm mgcl <dig>  1 m betaine , 1 μl of rnaseout  and  <dig> u of siii rt.
cycling conditions were 49 °c for 65 min, followed by 85 °c for 5 min.
two units of rnaseh  were then added to each reaction before a final incubation at 37 °c for  <dig> mins.
cdna samples were held at 12 °c before proceeding to the pcr reaction.table  <dig> primers for near full-length hcv amplification
afor degenerate primers, b = c or g or t, h = a or c or t, m = a or c, n = a or c or g or t, r = a or g, s = c or g, w = a or t, v = a or c or g, d = a or g or t, y = c or t
bgenome binding position with reference to hcv gt1a strain, h <dig>  genbank accession af009606
for optimisation experiments, the yield of near full-length amplicons was compared between two different superscript enzymes ii and iii  .
for the sii enzyme the rt reaction was performed as described above, except the superscript ii buffer and superscript ii enzyme was substituted into the assay.
jfh- <dig> rna was used as template.
comparison of different polymerase enzymes for efficient near full-length hcv amplification
using full-length jfh- <dig> cdna as template, transcribed as described above, two different polymerase combinations were tested for their efficiency in amplifying near full-length genome and a 4 kb fragment from the 5′ end of the hcv genome in a single round.
primers ky <dig> and vir45a  were used for near full-length amplification, while vir45a was replaced by hep <dig>  <cit>  for the 4 kb fragment.
the two enzymes tested in parallel were klentaq la  and klentaq dna polymerase  that was used at a ratio of  <dig> to  <dig> with pfu dna polymerase .
the reaction conditions of the klentaq la are detailed in the next paragraph.
the reaction conditions for the klentaq/pfu mix were performed as described by zhang et al.
<cit>  with the exception that the pcr cycling conditions were the same as those performed with the klentaq la reaction, as described below.
pcr of near full-length hcv genome
first round pcr was performed using 5 μl of cdna in a 50 μl pcr reaction containing 1× klentaq la buffer ,  <dig>  m betaine, 200 μm of each dntp,  <dig>  μm of the forward primer ky <dig>   <dig>  μm of the genotype specific reverse primer  and 1u of klentaq la enzyme.
pcr was performed at 94 °c for 2 min, 10 cycles at 94 °c for 30 s, 55 °c for 20 s, and 68 °c for 11 min, followed by an additional 20– <dig> cycles at 94 °c for 30 s, 57 °c for 20 s, and 68 °c for 10:30 min , with a final extension for 5 min at 68 °c.
the nested round of pcr was performed with 5 μl of first round product in a 50 μl reaction as described for the first round, except  <dig>  μm of hep21b was used as the forward primer and  <dig>  μm of a nested genotype-specific reverse primer .
pcr was performed at 94 °c for 2 min, 10 cycles at 94 °c for 30 s, 58 °c for 20 s, and 68 °c for 10:30 min, followed by an additional 20– <dig> cycles at 94 °c for 30 s, 60 °c for 20 s, and 68 °c for 10:30 min , and then followed by a final extension for 5 min at 68 °c.
following positive identification of a band of the correct size, which was approximately 9206 bp  , the products were purified using the agencourt ampure xp beads  according to the manufacturers instructions, eluted in nuclease-free water and stored at -80c.
real-time nested pcr
hcv rna was quantified by real-time pcr using a bio-rad myiq single-color real-time pcr detection system  as described previously  <cit>  with the exception that primers hep21b  and hep <dig> were used  <cit>  and near full-length cdna generated as described above, was used as template.
pcr band intensities were also quantified by densitometry using imagej .
illumina sequencing
libraries were prepared from the amplicon using either the nextera xt or truseq nano dna library prep kits  before sequencing using a miseq benchtop sequencer generating 2 × 300 bp length paired-end reads .
ngs reads were aligned using bowtie  <dig>  <cit>  implemented in geneious package version  <dig>  <cit> .
reads were aligned against a panel of full-length reference genomes that were obtained from genbank to represent gts 1– <dig> , and consensus sequences were generated from the aligned contigs.
mixed infection was identified by the generation of multiple contigs against two or more reference genomes.
in these situations, a consensus genome sequence was generated for each contig.fig.
<dig> phylogenetic analysis of the near full-length hcv genomes from  <dig> clinical samples.
the phylogenetic tree was generated using the neighbour-joining method implemented in geneious package version  <dig>  <cit> .
reference genomes are italicised with their subtype listed first, followed by their genbank accession number.
sequences that belonged to clinical samples with known mixed infections are underlined .
branches are colored according to hcv genotype.
the branch lengths are proportional to the evolutionary distance between sequences and the distance scale, in nucleotide substitutions per position, is shown
pacbio sequencing
one of the full-length amplicons, rimm, was selected for generating >9 kb sequence reads with pacbio smrt sequencing.
unique pacbio barcodes were ligated to the amplicon and approximately  <dig>  fmoles of the product was then sequenced via 240-min movies on one smrt cell using p6-c <dig> chemistry on a pacbio rs ii sequencer .
genome filtering, assembly and reassembly were performed using tools within smrt analysis v <dig> .
smrtbell adapter sequences were removed and circular consensus sequence  reads with a minimum of  <dig> full passes of the full amplicon  were selected for further analysis.
these reads were de novo assembled using vicuna  <cit> .
haplotype reconstruction of near full-length hcv quasispecies using pacbio smrt sequencing
pacbio rs ii reads were analysed using haplotype reconstruction analysis, as previously performed  <cit> .
briefly, haplotype analysis was performed to correct for random technical errors using the software shorah  <cit> .
only unique haplotypes and their estimated frequency of occurrence within the quasispecies population were reported.
indels identified in homopolymeric regions were manually replaced with the consensus sequence.
phylogenetic analysis
the  <dig> illumina generated consensus genomes from the  <dig> clinical samples and the cell culture derived test sample jfh- <dig> were aligned against representative subtypes from the gt <dig> to  <dig> reference genomes with the alignment tool muscle, implemented in geneious package version  <dig>  <cit> .
bootstrapped trees  were constructed using the neighbour-joining method, also implemented in geneious package version  <dig>  <cit> .
phylogenetic analysis with the pacbio generated haplotypes was performed as described above.
RESULTS
primer design
full-length genome alignments representing all six hcv gts were used to assess previously published and novel primer sites for pan-genotypic potential.
the previously published oligo da <dig> primer was selected for the rt step due to its pan-genotypic quality  <cit> .
it was possible to identify pan-genotypic primers for both the forward outer  and nested  primers, but genotype-specific primers were designed for the reverse primer pairs, outer and nested .
rna extraction optimisation
for the rna extraction, the qiamp viral rna mini kit was used but several modifications were made to the manufacturer’s protocol.
these modifications are outlined in detail in the methods section.
in brief, the carrier rna was substituted with 5 μg of linear acrylamide, as previously recommended for other rna viruses, hiv, rsv and wnv  <cit> .
elution of purified rna in rna storage solution was essential for optimal results, particularly after long-term storage and repeated freeze-thaw cycles.
successful amplification of near full-length genomes from stored rna samples was reproducible six months after initial extraction.
the centrifugation speeds were also reduced as this was shown to reduce shearing of rna.
also as successful amplification of the full genome requires intact genomes, mixing of sample by pipetting and vortexing was limited.
cdna optimisation
as part of the optimisation process for cdna generation, rt enzymes, reaction conditions and thermocycling conditions were examined using jfh- <dig> rna as template and near full-length amplicon yield as a measure of success.
for the rt enzyme analysis, two rt enzymes, superscript ii and iii were compared, with the siii enzyme generating a  <dig> -fold higher yield.
for the rt reaction conditions, it has been previously shown that the addition of betaine at 1 m concentration is optimal in similar assay conditions  <cit> .
therefore, we tested the effect of betaine at a final concentration of 1 m on amplicon yield.
the cdna reactions containing 1 m betaine generated an amplicon with a  <dig> -fold increase in yield compared to cdna conditions with no betaine .
in regard to thermocycling conditions, previous reports had indicated optimal cdna generation of full-length hcv with an extension time of  <dig>  h with varying extension temperatures  <cit> .
in our study, optimal results were obtained with a constant extension temperature of 49 °c for  <dig> mins.
it was also noted that more consistent results were obtained when the pcr proceeded immediately after the rt step had finished.
storage of cdna at either −20 °c or −80 °c greatly reduced the success rate in generating near full-length amplicons.
pcr optimisation
for pcr optimisation, two different klentaq mixes were compared.
the polymerase combination of klentaq and pfu at a ratio of  <dig> to  <dig>  as outlined by zhang et al.
<cit> , was compared using either the thermocyling conditions published by zhang et al.
or as recommended for the other commercial klentaq mix, klentaq la .
both of these conditions were used for the klentaq/pfu mix and compared directly to the commercial polymerase mix of klentaq la  that only used the conditions recommended by clontech.
the three different enzyme/assay conditions were tested for amplification efficiency using primer sets that would either generate a 9 kb or 4 kb fragment.
no amplicons for either the  <dig> or 9 kb amplicon were generated following the reaction and cycling conditions nominated by zhang et al.
<cit> .
for the remaining two enzyme/assay conditions, the yield of the ~9 kb fragment was increased by  <dig> -fold with the commercial mix, klentaq la compared to the klentaq/pfu mix run with the same cycling conditions .
a 4 kb region covering the 5′ end of the genome was also amplified with both polymerase mixes using clontech’s conditions and only a slight increase,  <dig> -fold, in amplicon yield of the 4 kb fragment was observed for the klentaq la polymerase mix.
overall, these results demonstrated the superior performance of the klentaq la mix for the near full-length amplicon, and the kit was adopted for further optimisation.
generally, the protocol provided with the klentaq la enzyme from clontech was found to be optimal with the only modification being the addition of betaine.
after the addition of 5 μl of cdna to the pcr assay the final betaine concentration was  <dig>  m, which is the recommended concentration for the klentaq enzyme.
the efficiency of amplicon generation, in terms of yield of amplicon and reduced non-specific amplification was improved if the rt and pcr primers were aliquoted into small batches with no subsequent freeze/thaw cycles before use.
freeze/thaw rounds of dntps did not appear to affect the generation of amplicons.
detection limit
to determine the detection limit of the nested pcr assay, a representative cdna sample for gt <dig>   <dig> and  <dig> was generated.
the copy number of near full-length cdna transcripts was determined by real-time pcr targeting the 5′utr of the genome.
the cdna samples were then serially diluted and the limit of detection assessed.
the assay had a detection limit of 1– <dig> copies of hcv near full-length cdna per reaction.
for this assay, we calculated the detection limit using near full-length cdna transcripts.
this was chosen as there are many independent variables that impact the generation of full-length cdna transcripts from a hcv positive plasma sample  <cit> .
for instance, plasma collection protocols, date of collection and storage conditions - particularly in regards to time delays post collection and freeze/thaw occurrence can result in rna degradation, and subsequently impact the generation of full-length cdna transcripts.
traditionally, methods used to determine viral rna genome copies target a small region of the genome and are resilient to rna degradation and therefore not a good indicator of the integrity of full length viral rna genomes.
robustness of near full-length amplification
to test the robustness of the assay on clinical samples,  <dig> hcv samples with known genotypes were tested for near full-length nested rt-pcr amplification.
the viral loads ranged from  <dig> to  <dig>  × 107 iu/ml.
the lowest viral load that was able to generate a near full-length amplicon was  <dig>  iu/ml.
all  <dig> samples with a viral load below  <dig>  iu/ml failed to amplify .
another  <dig> samples with viral loads above  <dig>  iu/ml also failed to amplify.
therefore, sensitivity testing indicated that the pcr was robust with  <dig>  % of the samples being successfully amplified with a viral load > <dig>  iu/ml  .
analysis of the data stratified by genotype, revealed that gt2b had the lowest success rate of  <dig>  %, followed by gt <dig> and gt <dig> at  <dig>  % and  <dig>  %, respectively .
all remaining genotype samples amplified with a 100 % success rate, although it should be noted that due to these other genotypes being less common in our cohorts their representation in the sample set was lower .
a representative set of amplicons  for each genotype were sequenced on the illumina miseq platform.
phylogenetic analysis was used to confirm the success of the assay in amplifying gts 1– <dig> .fig.
<dig> sensitivity of the near full-length assay tested on hcv positive clinical samples.
the success rate of amplifying near-full length genomes in relation to viral load is shown for  <dig> samples.
eighty-six of the  <dig> hcv positive samples with a viral load greater than  <dig>  iu/ml were successfully amplified .
all  <dig> samples with a viral load below  <dig>  iu/ml failed to amplify, however,  <dig> of these samples were retested following concentration by ultracentrifugation, of which three  were successfully amplified
2ab
aonly includes samples with viral loads greater than  <dig>  iu/ml
bthis was cell cultured derived jfh- <dig> and was not included in the success rate
ultracentrifugation
for samples with low viral loads  sensitivity of detection could be improved by concentrating the virus from the plasma by ultracentrifugation.
in this study we selected  <dig> samples that had detectable rna with the real-time pcr assay that targeted a 175 bp region in the 5′utr, but could not initially be amplified with the near full-length assay.
for these samples, rna was extracted from 1 ml of plasma after ultracentrifugation.
upon retesting, a near full-length amplicon was generated for  <dig> of the  <dig> samples that had previously failed.
while ultracentrifugation of larger sample volumes improved the sensitivity of near full-length amplification, we observed that simply increasing the plasma volume from which virus was extracted without ultracentrifugation did not.
we speculate that either the additional spin duration, total extraction time or additional pcr inhibitors from the increased plasma volume may account for the reduced rna extraction efficiency when the plasma volume extracted through the qiamp column is increased.
near full-length sequencing on pacbio
to test whether near full-length sequence reads could be generated from the hcv amplicon, the sample rimm was selected for sequencing on the pacbio rs ii.
a total of  <dig>  reads had a minimum of two full passes of the  <dig>  kb amplicon  and were used for further analysis.
the near-full length pacbio reads were then error corrected via haplotype reconstruction analysis  <cit> .
this analysis showed  <dig> distinct variants with the most common having a frequency of occurrence of 11 % , which was identical to the consensus genome sequence generated from the illumina data.fig.
<dig> analysis of the hcv quasispecies utilising near full-length reads with the pacbio rs platform.
individual sequences reads greater than 9 kb were generated and aligned for an hcv infected individual, rimm.
panel .
highlighter plot  representing  <dig> unique haplotypes that were detected from sequence reads.
the name of each haplotype shows the frequency of occurrence within the population .
the master sequence on the top of the plot is the consensus genome sequence obtained from the illumina sequences.
panel .
phylogenetic tree showing the genetic relationship between near-full length quasispecies obtained from pacbio reads
the sensitivity of pacbio to detect low frequency mutations was compared to the data generated by illumina from the same amplicon.
both sequencing platforms detected all  <dig> snps with a frequency greater than 7 % .
the pacbio reads after cleaning with shorah only detected  <dig>  %  of the snps detected with the illumina platform with a frequency below 7 %.
however, it should be noted that the genome sequence coverage generated by illumina was about five-fold higher, with a mean coverage of  <dig>  reads/site, respectively and likely accounts for the discrepancy in calling low frequency events.
phylogenetic analysis of the near-full length haplotypes was performed to understand the relationship of the quasispecies in the acutely infected hcv subject.
the analysis depicted the presence of two distinct sub-populations that carry most of their diversity in the structural region of the genome .
this analysis confirms the applicability of this approach to perform near-full genome quasispecies analysis.
multiple infection
due to the transmission dynamics of hcv, multiple infections can occur concurrently producing a “co-infection” with two or more hcv genotypes present at a single time point.
in the cohort used in this study the multiple infection rate has been previously reported to be as high as 25 %  <cit> .
we therefore selected two known multiple infection samples, a gt1a/3a and a gt1a/2b sample .
we multiplexed the reverse primers at equimolar concentrations to test if both genotypes could be simultaneously amplified in the same nrt-pcr reaction.
to confirm that this was successful the single amplicon was sequenced on the illumina platform and the sequence data aligned against both the gt reference sequences .
sequence reads were successfully mapped to both genotypes from the same sample.
the data for subject hitp <dig> is shown in fig.
<dig>  where  <dig>  % and  <dig>  % of sequence reads aligning to gt1a and 3a, respectively.
consensus sequences for both genotypes identified in each of the two subjects, hitp <dig> and hitp <dig> were generated and were clearly shown by phylogenetic analysis to belong to two separate genotypes .
the potential of this assay to be used to quantify mixed infection was not assessed.
we believe that differences in genotype-specific pcr efficiency due to pcr primer bias and differences in secondary rna structure would likely result in a semi-quantitative output.
in order to quantify co-infections, a rt-pcr targeting a smaller region and with higher sensitivity would be more reliable  <cit> .
the advantage of this assay is that it describes a pipeline for a cost efficient method to obtain full-genome sequences for phylogenetic and snp analysis without the need to duplicate pcr assays for multiple genotypes in a single infection.fig.
<dig> subject hitsp300345_ <dig> was identified as being infected with two hcv genotypes , gt1a and gt3a.
the hcv near full-length amplification protocol was adapted to amplify both subtypes in the same reaction and the amplicon was submitted for next generation sequencing with the miseq 2 ×  <dig> pe chemistry.
analysis of the aligned reads identified two populations, a gt1a and a gt3a population.
the presence of two hcv viral populations is shown in a coverage plot stratified by genome position
CONCLUSIONS
in this study we have described a robust assay that can amplify the near full-length genome from all six major hcv gts.
the method can also be simply adapted to detect and sequence multiple infection.
the ability to amplify the full genome in a single amplicon as opposed to multiple fragments reduces upstream cost and labour for multiple pcr reactions.
furthermore this method can be easily applied to sequence via multiple platforms, including sanger sequencing, single genome amplification, short read deep sequencing such as the illumina and roche platforms, or the long read platforms, such as pacbio.
as the sequence read technology continues to improve, the near-full length sequence data will improve analyses across an array of virological interests, including the ability to perform in-depth within-host evolutionary analysis and also to look at linkage between emerging antiviral resistant sites.
availability of supporting data
the genbank accession numbers for the near-full length genome illumina generated consensus nucleotide sequences of the samples in fig.
1 are as follows: ku <dig> to ku <dig>  kj <dig>  kj <dig> and kj <dig>
additional file
additional file 1: figure s <dig>  successful amplification of near full-length hcv genomes from multiple genotypes.
amplicons  were run on an agarose gel  and visualized on a gel doc molecular imager ; m represents the dna marker hyperladder  <dig> .
lanes  <dig> to  <dig> represent gt1a amplicons.
lanes  <dig> to  <dig> represent gt1b amplicons.
lanes  <dig> to  <dig> represent gt3a amplicons.
all amplicons were purified and successfully sequenced on the illumina platform, including the faintly visible band in lane  <dig>
abbreviations
daadirect acting antiviral
gtgenotype
hcvhepatitis c virus
ngsnext generation sequencing
nsnon-structural
pacbiopacific biosciences
pcrpolymerase chain reaction
rtreverse transcription
smrtsingle molecule real time
competing interests
at, ml, so and ch are full time employees of pacific biosciences, a company commercializing smrt sequencing technologies.
pacific biosciences provided the pacbio sequencing free of charge.
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interests.
authors’ contributions
rab and ae designed and conducted experiments, and wrote the manuscript.
rab, ae, cr, mrp, mw and smm performed experiments.
arl, lm and js provided samples.
bb-s and fl assisted with sequence analysis.
at, ml, so and ch performed the pacbio sequencing.
all authors have read and approved the manuscript.

 hepatitis c virus is a significant human pathogen affecting nearly 3 % of the world’s population, and is a leading cause of chronic liver diseases including cirrhosis and hepatocellular carcinoma. hcv is a member of the flaviviridae family and has a single stranded rna genome that is  kb in length with positive polarity. the genome contains a single open-reading frame and encodes a precursor polyprotein of approximately amino acid residues. within an infected cell, this polyprotein is processed by cellular and host proteases to yield ten structural and non-structural proteins. the viral rna-dependent rna polymerase, or ns5b, is a key enzyme in the hcv replication complex within an infected cell, and is responsible for the production of nascent genomes for packaging into new virions. the polymerase is highly error-prone, a feature common to many rna viruses, and as a result hcv exists as seven distinct genotypes, which differ by up to 35 % at the nucleotide level. within each genotype, viruses have been further classified into subtypes with about 20 % inter-subtype nucleotide divergence. the genotypes are unevenly distributed across the globe, with genotypes and being the most prevalent, and genotype being the rarest having only been detected once in canada from a central african immigrant. with the advent of direct-acting antivirals for the treatment of hcv infections, there is a need to monitor the emergence of resistance-associated variants before and after treatment. while testing specific regions of the hcv genome using both consensus and next generation sequencing has enabled such monitoring in daa therapies, including agents targeting ns ns5a and ns5b, the emerging daa combination regimens emphasise the necessity to simultaneously screen multiple genes of the viral genome in a simple, cost-effective manner. furthermore, there is a need to detect whether individual viral variants carry multiple polymorphisms conferring resistance against all daas in a combination regimen, therefore increasing the chances of viral persistence against such therapies. in the past, such analysis was possible only by cloning and consensus sequencing. however, as the ngs technologies continue to improve read length the range for covariant studies continues to increase. in addition to monitoring viral variants associated with drug resistance, methods for the molecular amplification and sequencing of hcv rna have also been instrumental in characterising hcv infections, including studies seeking to understand virus transmission and within-host evolution. previously, these methods focused on specific regions of the hcv genome, or analysed the entire genome in separate fragments, which is laborious, cost-prohibitive and leads to analysis issues, including uneven coverage due to amplicon pooling, artificial recombinants during genome assemby and multiple pcr primer bias. therefore, there is a need for sensitive methods that can amplify the entire viral genome from all gts in clinical samples. a few reports have described the amplification of near full-length hcv genomes, however these were either limited by the sensitivity of the methodology or coverage of all major genotypes. recently, rna sequencing technology has been applied to sequence hcv in a non-specific manner. while these methods offer the advantage of reduced primer bias and reduced upstream labour, they require increased labour in data analysis, have reduced sensitivity and increased overall cost per sample. here, we describe a simple method which allows sensitive amplification of near full-length hcv genomes from gt to using this method, 90 % of a set of hcv-infected clinical samples were successfully amplified and sequenced using an ngs approach. as a proof of principle, the method was also applied to amplify and sequence near full-length hcv genomes from two subjects co-infected with multiple genotypes. the ability to generate near full-length quasispecies sequence was tested with one amplicon using the pacbio rs ii platform. methods cohort stored plasma samples positive for hcv gt and were made available from the hepatitis c incidence and transmission study in prisons and community cohort, which is a prospective cohort of hcv seronegative and hcv rna negative subjects in new south wales, australia. stored plasma samples positive for hcv gt and were from academisch medisch centrum patients identified with hcv infection. in all subjects, hcv infection was confirmed by detection of hcv-specific antibody and rna. in the hits cohort, hcv antibody testing was performed using the qualitative abbott architect anti-hcv chemiluminescent microparticle immunoassay. for the amc patients hcv antibody testing was performed using the axsym hcv serology test. for all subjects quantitative hcv rna detection was performed with the cobas ampliprep/cobas taqman hcv assay. ethics statement for the hits samples ethical approvals were obtained from human research ethics committees of justice health, new south wales department of corrective services, and the university of new south wales, all located in sydney, australia. written informed consent was obtained from the participants. for the dutch samples, the study was performed according to the dutch federa code of conduct for responsible use of human tissue and medical research genotyping and detection of multiple infection genotype determination and detection of multiple hcv genotype infection was performed on a region of the core as previously described. primer design full-length genome sequences representing each of the six hcv genotypes were downloaded from genbank and used for primer design. this included and different strains for gts 1– respectively. to choose primer binding sites we manually scanned the 3′ end of the genome for regions >20 bp with a minimum of 90 % identity. to improve sequence identity of the primer with the viral variants, degenerates bases were inserted where needed. to improve binding efficiency degenerate bases were not added to the last nucleotides at the 3′ end of the primer and where possible primers had c’s or g’s at the 3′ base. jfh- rna t transcripts t rna transcripts for full-length cell culture derived hcv genotype 2a variant, jfh- were generated as previously described. rna concentration by ultracentrifugation for concentration of hcv from plasma samples, –1 ml of plasma was thawed and centrifuged at  g for 10 min at 4 °c. supernatants were transferred and centrifuged at  g for  h at 4 °c after which the pellet was re-suspended in 140 μl of 1× pbs. viral rna was then extracted from the sample as outlined below. rna extraction viral rna was extracted from 140 μl of plasma using the qiamp viral rna kit according to manufacturers’ instructions, with the following modifications: ambion® linear acrylamide was used instead of the carrier rna provided in the kit; sample lysis was performed by inverting tubes instead of vortexing; and the speed of centrifugation was reduced to  g for all steps except the final wash which was carried out at  g. finally, rna was eluted in 50 μl of rna storage solution and stored at −80 °c. reverse transcription near full-length hcv cdna was synthesized from viral rna using the superscript iii first-strand synthesis system and a pan-genotype primer. before commencing reverse-transcription, 7 μl of rna, 1 μl of 10 μm primer and 1 μl of 10 mm dntps were mixed and incubated in a thermocycler at 65 °c for mins and then placed immediately on ice for 1 min. rt was then initiated with the addition of rt reaction mix to a 20 μl final volume at a final concentration 1x rt buffer, 5 mm mgcl 1 m betaine, 1 μl of rnaseout and u of siii rt. cycling conditions were 49 °c for 65 min, followed by 85 °c for 5 min. two units of rnaseh were then added to each reaction before a final incubation at 37 °c for mins. cdna samples were held at 12 °c before proceeding to the pcr reaction.table primers for near full-length hcv amplification afor degenerate primers, b = c or g or t, h = a or c or t, m = a or c, n = a or c or g or t, r = a or g, s = c or g, w = a or t, v = a or c or g, d = a or g or t, y = c or t bgenome binding position with reference to hcv gt1a strain, h genbank accession af009606 for optimisation experiments, the yield of near full-length amplicons was compared between two different superscript enzymes ii and iii. for the sii enzyme the rt reaction was performed as described above, except the superscript ii buffer and superscript ii enzyme was substituted into the assay. jfh- rna was used as template. comparison of different polymerase enzymes for efficient near full-length hcv amplification using full-length jfh- cdna as template, transcribed as described above, two different polymerase combinations were tested for their efficiency in amplifying near full-length genome and a 4 kb fragment from the 5′ end of the hcv genome in a single round. primers ky and vir45a were used for near full-length amplification, while vir45a was replaced by hep for the 4 kb fragment. the two enzymes tested in parallel were klentaq la and klentaq dna polymerase that was used at a ratio of to with pfu dna polymerase. the reaction conditions of the klentaq la are detailed in the next paragraph. the reaction conditions for the klentaq/pfu mix were performed as described by zhang et al. with the exception that the pcr cycling conditions were the same as those performed with the klentaq la reaction, as described below. pcr of near full-length hcv genome first round pcr was performed using 5 μl of cdna in a 50 μl pcr reaction containing 1× klentaq la buffer,  m betaine, 200 μm of each dntp,  μm of the forward primer ky  μm of the genotype specific reverse primer and 1u of klentaq la enzyme. pcr was performed at 94 °c for 2 min, 10 cycles at 94 °c for 30 s, 55 °c for 20 s, and 68 °c for 11 min, followed by an additional 20– cycles at 94 °c for 30 s, 57 °c for 20 s, and 68 °c for 10:30 min, with a final extension for 5 min at 68 °c. the nested round of pcr was performed with 5 μl of first round product in a 50 μl reaction as described for the first round, except  μm of hep21b was used as the forward primer and  μm of a nested genotype-specific reverse primer. pcr was performed at 94 °c for 2 min, 10 cycles at 94 °c for 30 s, 58 °c for 20 s, and 68 °c for 10:30 min, followed by an additional 20– cycles at 94 °c for 30 s, 60 °c for 20 s, and 68 °c for 10:30 min, and then followed by a final extension for 5 min at 68 °c. following positive identification of a band of the correct size, which was approximately 9206 bp, the products were purified using the agencourt ampure xp beads according to the manufacturers instructions, eluted in nuclease-free water and stored at -80c. real-time nested pcr hcv rna was quantified by real-time pcr using a bio-rad myiq single-color real-time pcr detection system as described previously with the exception that primers hep21b and hep were used and near full-length cdna generated as described above, was used as template. pcr band intensities were also quantified by densitometry using imagej. illumina sequencing libraries were prepared from the amplicon using either the nextera xt or truseq nano dna library prep kits before sequencing using a miseq benchtop sequencer generating 2 × 300 bp length paired-end reads. ngs reads were aligned using bowtie implemented in geneious package version. reads were aligned against a panel of full-length reference genomes that were obtained from genbank to represent gts 1–, and consensus sequences were generated from the aligned contigs. mixed infection was identified by the generation of multiple contigs against two or more reference genomes. in these situations, a consensus genome sequence was generated for each contig.fig. phylogenetic analysis of the near full-length hcv genomes from clinical samples. the phylogenetic tree was generated using the neighbour-joining method implemented in geneious package version. reference genomes are italicised with their subtype listed first, followed by their genbank accession number. sequences that belonged to clinical samples with known mixed infections are underlined. branches are colored according to hcv genotype. the branch lengths are proportional to the evolutionary distance between sequences and the distance scale, in nucleotide substitutions per position, is shown pacbio sequencing one of the full-length amplicons, rimm, was selected for generating >9 kb sequence reads with pacbio smrt sequencing. unique pacbio barcodes were ligated to the amplicon and approximately fmoles of the product was then sequenced via 240-min movies on one smrt cell using p6-c chemistry on a pacbio rs ii sequencer. genome filtering, assembly and reassembly were performed using tools within smrt analysis v. smrtbell adapter sequences were removed and circular consensus sequence reads with a minimum of full passes of the full amplicon were selected for further analysis. these reads were de novo assembled using vicuna. haplotype reconstruction of near full-length hcv quasispecies using pacbio smrt sequencing pacbio rs ii reads were analysed using haplotype reconstruction analysis, as previously performed. briefly, haplotype analysis was performed to correct for random technical errors using the software shorah. only unique haplotypes and their estimated frequency of occurrence within the quasispecies population were reported. indels identified in homopolymeric regions were manually replaced with the consensus sequence. phylogenetic analysis the illumina generated consensus genomes from the clinical samples and the cell culture derived test sample jfh- were aligned against representative subtypes from the gt to reference genomes with the alignment tool muscle, implemented in geneious package version. bootstrapped trees were constructed using the neighbour-joining method, also implemented in geneious package version. phylogenetic analysis with the pacbio generated haplotypes was performed as described above.  primer design full-length genome alignments representing all six hcv gts were used to assess previously published and novel primer sites for pan-genotypic potential. the previously published oligo da primer was selected for the rt step due to its pan-genotypic quality. it was possible to identify pan-genotypic primers for both the forward outer and nested  primers, but genotype-specific primers were designed for the reverse primer pairs, outer and nested. rna extraction optimisation for the rna extraction, the qiamp viral rna mini kit was used but several modifications were made to the manufacturer’s protocol. these modifications are outlined in detail in the methods section. in brief, the carrier rna was substituted with 5 μg of linear acrylamide, as previously recommended for other rna viruses, hiv, rsv and wnv. elution of purified rna in rna storage solution was essential for optimal results, particularly after long-term storage and repeated freeze-thaw cycles. successful amplification of near full-length genomes from stored rna samples was reproducible six months after initial extraction. the centrifugation speeds were also reduced as this was shown to reduce shearing of rna. also as successful amplification of the full genome requires intact genomes, mixing of sample by pipetting and vortexing was limited. cdna optimisation as part of the optimisation process for cdna generation, rt enzymes, reaction conditions and thermocycling conditions were examined using jfh- rna as template and near full-length amplicon yield as a measure of success. for the rt enzyme analysis, two rt enzymes, superscript ii and iii were compared, with the siii enzyme generating a -fold higher yield. for the rt reaction conditions, it has been previously shown that the addition of betaine at 1 m concentration is optimal in similar assay conditions. therefore, we tested the effect of betaine at a final concentration of 1 m on amplicon yield. the cdna reactions containing 1 m betaine generated an amplicon with a -fold increase in yield compared to cdna conditions with no betaine. in regard to thermocycling conditions, previous reports had indicated optimal cdna generation of full-length hcv with an extension time of  h with varying extension temperatures. in our study, optimal results were obtained with a constant extension temperature of 49 °c for mins. it was also noted that more consistent results were obtained when the pcr proceeded immediately after the rt step had finished. storage of cdna at either −20 °c or −80 °c greatly reduced the success rate in generating near full-length amplicons. pcr optimisation for pcr optimisation, two different klentaq mixes were compared. the polymerase combination of klentaq and pfu at a ratio of to as outlined by zhang et al., was compared using either the thermocyling conditions published by zhang et al. or as recommended for the other commercial klentaq mix, klentaq la. both of these conditions were used for the klentaq/pfu mix and compared directly to the commercial polymerase mix of klentaq la that only used the conditions recommended by clontech. the three different enzyme/assay conditions were tested for amplification efficiency using primer sets that would either generate a 9 kb or 4 kb fragment. no amplicons for either the or 9 kb amplicon were generated following the reaction and cycling conditions nominated by zhang et al.. for the remaining two enzyme/assay conditions, the yield of the ~9 kb fragment was increased by -fold with the commercial mix, klentaq la compared to the klentaq/pfu mix run with the same cycling conditions. a 4 kb region covering the 5′ end of the genome was also amplified with both polymerase mixes using clontech’s conditions and only a slight increase, -fold, in amplicon yield of the 4 kb fragment was observed for the klentaq la polymerase mix. overall, these results demonstrated the superior performance of the klentaq la mix for the near full-length amplicon, and the kit was adopted for further optimisation. generally, the protocol provided with the klentaq la enzyme from clontech was found to be optimal with the only modification being the addition of betaine. after the addition of 5 μl of cdna to the pcr assay the final betaine concentration was  m, which is the recommended concentration for the klentaq enzyme. the efficiency of amplicon generation, in terms of yield of amplicon and reduced non-specific amplification was improved if the rt and pcr primers were aliquoted into small batches with no subsequent freeze/thaw cycles before use. freeze/thaw rounds of dntps did not appear to affect the generation of amplicons. detection limit to determine the detection limit of the nested pcr assay, a representative cdna sample for gt and was generated. the copy number of near full-length cdna transcripts was determined by real-time pcr targeting the 5′utr of the genome. the cdna samples were then serially diluted and the limit of detection assessed. the assay had a detection limit of 1– copies of hcv near full-length cdna per reaction. for this assay, we calculated the detection limit using near full-length cdna transcripts. this was chosen as there are many independent variables that impact the generation of full-length cdna transcripts from a hcv positive plasma sample. for instance, plasma collection protocols, date of collection and storage conditions - particularly in regards to time delays post collection and freeze/thaw occurrence can result in rna degradation, and subsequently impact the generation of full-length cdna transcripts. traditionally, methods used to determine viral rna genome copies target a small region of the genome and are resilient to rna degradation and therefore not a good indicator of the integrity of full length viral rna genomes. robustness of near full-length amplification to test the robustness of the assay on clinical samples, hcv samples with known genotypes were tested for near full-length nested rt-pcr amplification. the viral loads ranged from to × 107 iu/ml. the lowest viral load that was able to generate a near full-length amplicon was  iu/ml. all samples with a viral load below  iu/ml failed to amplify. another samples with viral loads above  iu/ml also failed to amplify. therefore, sensitivity testing indicated that the pcr was robust with  % of the samples being successfully amplified with a viral load >  iu/ml. analysis of the data stratified by genotype, revealed that gt2b had the lowest success rate of  %, followed by gt and gt at  % and  %, respectively. all remaining genotype samples amplified with a 100 % success rate, although it should be noted that due to these other genotypes being less common in our cohorts their representation in the sample set was lower. a representative set of amplicons for each genotype were sequenced on the illumina miseq platform. phylogenetic analysis was used to confirm the success of the assay in amplifying gts 1–.fig. sensitivity of the near full-length assay tested on hcv positive clinical samples. the success rate of amplifying near-full length genomes in relation to viral load is shown for samples. eighty-six of the hcv positive samples with a viral load greater than  iu/ml were successfully amplified. all samples with a viral load below  iu/ml failed to amplify, however, of these samples were retested following concentration by ultracentrifugation, of which three were successfully amplified 2ab aonly includes samples with viral loads greater than  iu/ml bthis was cell cultured derived jfh- and was not included in the success rate ultracentrifugation for samples with low viral loads sensitivity of detection could be improved by concentrating the virus from the plasma by ultracentrifugation. in this study we selected samples that had detectable rna with the real-time pcr assay that targeted a 175 bp region in the 5′utr, but could not initially be amplified with the near full-length assay. for these samples, rna was extracted from 1 ml of plasma after ultracentrifugation. upon retesting, a near full-length amplicon was generated for of the samples that had previously failed. while ultracentrifugation of larger sample volumes improved the sensitivity of near full-length amplification, we observed that simply increasing the plasma volume from which virus was extracted without ultracentrifugation did not. we speculate that either the additional spin duration, total extraction time or additional pcr inhibitors from the increased plasma volume may account for the reduced rna extraction efficiency when the plasma volume extracted through the qiamp column is increased. near full-length sequencing on pacbio to test whether near full-length sequence reads could be generated from the hcv amplicon, the sample rimm was selected for sequencing on the pacbio rs ii. a total of reads had a minimum of two full passes of the  kb amplicon and were used for further analysis. the near-full length pacbio reads were then error corrected via haplotype reconstruction analysis. this analysis showed distinct variants with the most common having a frequency of occurrence of 11 %, which was identical to the consensus genome sequence generated from the illumina data.fig. analysis of the hcv quasispecies utilising near full-length reads with the pacbio rs platform. individual sequences reads greater than 9 kb were generated and aligned for an hcv infected individual, rimm. panel. highlighter plot representing unique haplotypes that were detected from sequence reads. the name of each haplotype shows the frequency of occurrence within the population. the master sequence on the top of the plot is the consensus genome sequence obtained from the illumina sequences. panel. phylogenetic tree showing the genetic relationship between near-full length quasispecies obtained from pacbio reads the sensitivity of pacbio to detect low frequency mutations was compared to the data generated by illumina from the same amplicon. both sequencing platforms detected all snps with a frequency greater than 7 %. the pacbio reads after cleaning with shorah only detected  % of the snps detected with the illumina platform with a frequency below 7 %. however, it should be noted that the genome sequence coverage generated by illumina was about five-fold higher, with a mean coverage of reads/site, respectively and likely accounts for the discrepancy in calling low frequency events. phylogenetic analysis of the near-full length haplotypes was performed to understand the relationship of the quasispecies in the acutely infected hcv subject. the analysis depicted the presence of two distinct sub-populations that carry most of their diversity in the structural region of the genome. this analysis confirms the applicability of this approach to perform near-full genome quasispecies analysis. multiple infection due to the transmission dynamics of hcv, multiple infections can occur concurrently producing a “co-infection” with two or more hcv genotypes present at a single time point. in the cohort used in this study the multiple infection rate has been previously reported to be as high as 25 %. we therefore selected two known multiple infection samples, a gt1a/3a and a gt1a/2b sample. we multiplexed the reverse primers at equimolar concentrations to test if both genotypes could be simultaneously amplified in the same nrt-pcr reaction. to confirm that this was successful the single amplicon was sequenced on the illumina platform and the sequence data aligned against both the gt reference sequences. sequence reads were successfully mapped to both genotypes from the same sample. the data for subject hitp is shown in fig.  where  % and  % of sequence reads aligning to gt1a and 3a, respectively. consensus sequences for both genotypes identified in each of the two subjects, hitp and hitp were generated and were clearly shown by phylogenetic analysis to belong to two separate genotypes. the potential of this assay to be used to quantify mixed infection was not assessed. we believe that differences in genotype-specific pcr efficiency due to pcr primer bias and differences in secondary rna structure would likely result in a semi-quantitative output. in order to quantify co-infections, a rt-pcr targeting a smaller region and with higher sensitivity would be more reliable. the advantage of this assay is that it describes a pipeline for a cost efficient method to obtain full-genome sequences for phylogenetic and snp analysis without the need to duplicate pcr assays for multiple genotypes in a single infection.fig. subject hitsp300345_ was identified as being infected with two hcv genotypes, gt1a and gt3a. the hcv near full-length amplification protocol was adapted to amplify both subtypes in the same reaction and the amplicon was submitted for next generation sequencing with the miseq 2 ×  pe chemistry. analysis of the aligned reads identified two populations, a gt1a and a gt3a population. the presence of two hcv viral populations is shown in a coverage plot stratified by genome position  in this study we have described a robust assay that can amplify the near full-length genome from all six major hcv gts. the method can also be simply adapted to detect and sequence multiple infection. the ability to amplify the full genome in a single amplicon as opposed to multiple fragments reduces upstream cost and labour for multiple pcr reactions. furthermore this method can be easily applied to sequence via multiple platforms, including sanger sequencing, single genome amplification, short read deep sequencing such as the illumina and roche platforms, or the long read platforms, such as pacbio. as the sequence read technology continues to improve, the near-full length sequence data will improve analyses across an array of virological interests, including the ability to perform in-depth within-host evolutionary analysis and also to look at linkage between emerging antiviral resistant sites. availability of supporting data the genbank accession numbers for the near-full length genome illumina generated consensus nucleotide sequences of the samples in fig. 1 are as follows: ku to ku kj kj and kj additional file additional file 1: figure s successful amplification of near full-length hcv genomes from multiple genotypes. amplicons were run on an agarose gel and visualized on a gel doc molecular imager; m represents the dna marker hyperladder. lanes to represent gt1a amplicons. lanes to represent gt1b amplicons. lanes to represent gt3a amplicons. all amplicons were purified and successfully sequenced on the illumina platform, including the faintly visible band in lane abbreviations daadirect acting antiviral gtgenotype hcvhepatitis c virus ngsnext generation sequencing nsnon-structural pacbiopacific biosciences pcrpolymerase chain reaction rtreverse transcription smrtsingle molecule real time competing interests at, ml, so and ch are full time employees of pacific biosciences, a company commercializing smrt sequencing technologies. pacific biosciences provided the pacbio sequencing free of charge. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interests. authors’ contributions rab and ae designed and conducted experiments, and wrote the manuscript. rab, ae, cr, mrp, mw and smm performed experiments. arl, lm and js provided samples. bb-s and fl assisted with sequence analysis. at, ml, so and ch performed the pacbio sequencing. all authors have read and approved the manuscript. 
we applied this protocol to hcv positive clinical samples, and had a successful genome amplification rate of 90 %, when the viral load was greater than  iu/ml. illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as hcv co-infection with multiple genotypes. all hcv genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. in this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major hcv genotypes. the assay was shown to have a detection limit of 1– cdna copies per reaction. hepatitis c virus is a rapidly evolving rna virus that has been classified into seven genotypes. although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient.
 hepatitis c virus is a rapidly evolving rna virus that has been classified into seven genotypes. all hcv genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. the genotypes are unevenly distributed across the globe, with genotypes and being the most prevalent. until recently, molecular epidemiological studies of hcv evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the  kb hcv genome. although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient.  in this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major hcv genotypes. we applied this protocol to hcv positive clinical samples, and had a successful genome amplification rate of 90 %, when the viral load was greater than  iu/ml. the assay was shown to have a detection limit of 1– cdna copies per reaction. the method was tested with both illumina and pacbio single molecule, real-time sequencing technologies. illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as hcv co-infection with multiple genotypes. the application of the method with pacbio rs resulted in sequence reads greater than 9 kb that covered the near full-length hcv amplicon in a single read and enabled analysis of the near full-length quasispecies.  the protocol described herein can be utilised for rapid amplification and sequencing of the near-full length hcv genome in a cost efficient manner suitable for a wide range of applications. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. http://dx.doi.org/ /501100000925national health and medical research council1042090rg1084706rg121994bull rowena a. lloyd andrew r. issue-copyright-statement© the author 2016 