title: Ultra-deep pyrosequencing (UDPS) data treatment to study amplicon HCV minor variants.
abstract: We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain ; Roche Diagnostics SL, Sant Cugat del Vallès, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Gregori
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain ; CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain ; Universitat Autònoma de Barcelona, Bellaterra, Spain.
authors.forename: Juan I
authors.initials: JI
authors.lastname: Esteban
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain ; CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Cubero
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain ; CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Damir
authors.initials: D
authors.lastname: Garcia-Cehic
authors.affiliation: CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain ; Centro de Biología Molecular Severo Ochoa (CBM), UAM, Madrid, Spain.
authors.forename: Celia
authors.initials: C
authors.lastname: Perales
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain ; Biochemistry Unit, HUVH, Barcelona, Spain.
authors.forename: Rosario
authors.initials: R
authors.lastname: Casillas
authors.affiliation: Roche Diagnostics SL, Sant Cugat del Vallès, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Alvarez-Tejado
authors.affiliation: CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain ; Universitat Autònoma de Barcelona, Bellaterra, Spain ; Biochemistry Unit, HUVH, Barcelona, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Rodríguez-Frías
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain ; CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain ; Universitat Autònoma de Barcelona, Bellaterra, Spain.
authors.forename: Jaume
authors.initials: J
authors.lastname: Guardia
authors.affiliation: CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain ; Centro de Biología Molecular Severo Ochoa (CBM), UAM, Madrid, Spain.
authors.forename: Esteban
authors.initials: E
authors.lastname: Domingo
authors.affiliation: Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain ; CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain ; Universitat Autònoma de Barcelona, Bellaterra, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Quer
mesh_terms: D003001:Cloning, Molecular; D024882:Drug Resistance, Viral / Q000235:genetics; D014644:Genetic Variation; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing / Q000706:statistics & numerical data*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000706:statistics & numerical data*; D017361:Viral Nonstructural Proteins / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase