Journal Article

VirVarSeq: a low-frequency virus variant detection pipeline for Illumina sequencing using adaptive base-calling accuracy filtering

Bie M.P. Verbist, Kim Thys, Joke Reumers, Yves Wetzels, Koen Van der Borght, Willem Talloen, Jeroen Aerssens, Lieven Clement and Olivier Thas

in Bioinformatics

Volume 31, issue 1, pages 94-101
Published in print January 2015 | ISSN: 1367-4803
Published online August 2014 | e-ISSN: 1460-2059 | DOI:
VirVarSeq: a low-frequency virus variant detection pipeline for Illumina sequencing using adaptive base-calling accuracy filtering

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Motivation: 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%.

Availability: The VirVarSeq is available, together with a user’s guide and test data, at sourceforge:


Supplementary information: Supplementary data are available at Bioinformatics online.

Journal Article.  6267 words.  Illustrated.

Subjects: Bioinformatics and Computational Biology

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