title: VirVarSeq: a low-frequency virus variant detection pipeline for Illumina sequencing using adaptive base-calling accuracy filtering.
abstract: BACKGROUND
  In virology, massively parallel sequencing (MPS) opens many opportunities for studying viral quasi-species, e.g. in HIV-1- and HCV-infected patients. This is essential for understanding pathways to resistance, which can substantially improve treatment. Although MPS platforms allow in-depth characterization of sequence variation, their measurements still involve substantial technical noise. For Illumina sequencing, single base substitutions are the main error source and impede powerful assessment of low-frequency mutations. Fortunately, base calls are complemented with quality scores (Qs) that are useful for differentiating errors from the real low-frequency mutations.
  
  
  RESULTS
  A variant calling tool, Q-cpileup, is proposed, which exploits the Qs of nucleotides in a filtering strategy to increase specificity. The tool is imbedded in an open-source pipeline, VirVarSeq, which allows variant calling starting from fastq files. Using both plasmid mixtures and clinical samples, we show that Q-cpileup is able to reduce the number of false-positive findings. The filtering strategy is adaptive and provides an optimized threshold for individual samples in each sequencing run. Additionally, linkage information is kept between single-nucleotide polymorphisms as variants are called at the codon level. This enables virologists to have an immediate biological interpretation of the reported variants with respect to their antiviral drug responses. A comparison with existing SNP caller tools reveals that calling variants at the codon level with Q-cpileup results in an outstanding sensitivity while maintaining a good specificity for variants with frequencies down to 0.5%.
  
  
  BACKGROUND
  The VirVarSeq is available, together with a user's guide and test data, at sourceforge: http://sourceforge.net/projects/virtools/?source=directory.
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Bie M P
authors.initials: BM
authors.lastname: Verbist
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Kim
authors.initials: K
authors.lastname: Thys
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Joke
authors.initials: J
authors.lastname: Reumers
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Yves
authors.initials: Y
authors.lastname: Wetzels
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Koen
authors.initials: K
authors.lastname: Van der Borght
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Willem
authors.initials: W
authors.lastname: Talloen
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Aerssens
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Lieven
authors.initials: L
authors.lastname: Clement
authors.affiliation: Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Janssen R&D, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Applied Mathematics, Informatics and Statistics, Ghent University, Krijgslaan 281 S9, 9000 Gent, Belgium and University of Wollongong, National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, NSW 2522, Australia.
authors.forename: Olivier
authors.initials: O
authors.lastname: Thas
mesh_terms: D000465:Algorithms; D014644:Genetic Variation / Q000235:genetics*; D016679:Genome, Viral; D023281:Genomics / Q000379:methods*; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000235:genetics* / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D012984:Software