Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants
Gregori i Font, Josep (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Esteban Mur, Juan Ignacio (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Cubero, María Dolores (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Garcia-Cehic, D. (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Perales Viejo, Celia (Centro de Biología Molecular Severo Ochoa)
Casillas, Rosario (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Alvarez-Tejado, Miguel (Roche Diagnostics, Sant Cugat del Vallès)
Rodríguez Frías, Francisco (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Guardia Massó, Jaume (Universitat Autònoma de Barcelona)
Domingo, Esteban (Centro de Biología Molecular Severo Ochoa)
Quer, Josep 1963- (Universitat Autònoma de Barcelona)

Data:	2013
Resum:	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.
Ajuts:	Ministerio de Ciencia e Innovación IDI-20110115 Ministerio de Ciencia e Innovación SAF 2009-10403 Instituto de Salud Carlos III PI10/01505 Ministerio de Ciencia e Innovación BFU2011-23604
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Publicat a:	PloS one, Vol. 8 (december 2013) , ISSN 1932-6203

DOI: 10.1371/journal.pone.0083361
PMID: 24391758

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