title: Near full length hepatitis C virus genome reconstruction by next generation sequencing based on genotype-independent amplification.
abstract: BACKGROUND
  Deep sequencing has a deep impact on the study of rapidly mutating RNA viruses, such as hepatitis C virus, proving to be an invaluable tool for analyzing virus diversity and evolution.
  
  
  OBJECTIVE
  Genotype-independent high-throughput pyrosequencing was used to obtain near full length hepatitis C virus genome sequence reconstruction directly from clinical samples.
  
  
  METHODS
  Samples from hepatitis C virus infected subjects harbouring different subtypes (1a, 1b, 2c) were analyzed (viral load range: 1.2-20.8 × 10(6)IU/ml). Data were generated with a modified sequence-independent single primer amplification method followed by 454 sequencing.
  
  
  RESULTS
  the extent of reconstructed hepatitis C virus genome varied from 79.95% to 99.64%. No correlation between extent of genome reconstruction and either viral load (r=0.4857, p=0.3556) or number of HCV reads (r=0.08571, p=0.9194) was observed.
  
  
  CONCLUSIONS
  This study describes a protocol for obtaining whole genome sequences from different hepatitis C virus patients with different genotypes in a single sequencing run.
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bartolini
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Giombini
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Abbate
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Selleri
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Rozera
authors.affiliation: University of Rome "Tor Vergata", Department of Biology, Rome, Italy.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Biagini
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy.
authors.forename: Ubaldo
authors.initials: U
authors.lastname: Visco-Comandini
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Taibi
authors.affiliation: National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy. Electronic address: maria.capobianchi@inmi.it.
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Capobianchi
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D016679:Genome, Viral; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010802:Phylogeny; D012367:RNA, Viral / Q000302:isolation & purification; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral
keywords: DAAs; Deep-sequencing; Full genome reconstruction; HCV; Modified SISPA approach