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Genetic Diversity and Evolution | Spotlight

The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies

Romain Gallet, Frédéric Fabre, Yannis Michalakis, Stéphane Blanc
Anne E. Simon, Editor
Romain Gallet
aINRA, UMR BGPI, INRA-CIRAD-SupAgro, Cirad TA-A54/K, Campus International de Baillarguet, Montpellier, France
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Frédéric Fabre
bUMR 1065 Santé et Agroécologie du Vignoble, INRA, Villenave d'Ornon, France
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Yannis Michalakis
cUMR MIVEGEC 5290, CNRS-IRD-Université de Montpellier, IRD, Montpellier, France
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Stéphane Blanc
aINRA, UMR BGPI, INRA-CIRAD-SupAgro, Cirad TA-A54/K, Campus International de Baillarguet, Montpellier, France
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Anne E. Simon
University of Maryland, College Park
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DOI: 10.1128/JVI.00561-17
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ABSTRACT

The invention of next-generation sequencing (NGS) techniques marked the coming of a new era in the detection of the genetic diversity of intrahost viral populations. A good understanding of the genetic structure of these populations requires, first, the ability to identify the different isolates or variants and, second, the ability to accurately quantify them. However, the initial amplification step of NGS studies can impose potential quantitative biases, modifying the variant relative frequencies. In particular, the number of target molecules (NTM) used during the amplification step is vastly overlooked although of primary importance, as it sets the limit of the accuracy and sensitivity of the sequencing procedure. In the present article, we investigated quantitative biases in an NGS study of populations of a multipartite single-stranded DNA (ssDNA) virus at different steps of the procedure. We studied 20 independent populations of the ssDNA virus faba bean necrotic stunt virus (FBNSV) in two host plants, Vicia faba and Medicago truncatula. FBNSV is a multipartite virus composed of eight genomic segments, whose specific and host-dependent relative frequencies are defined as the “genome formula.” Our results show a significant distortion of the FBNSV genome formula after the amplification and sequencing steps. We also quantified the genetic bottleneck occurring at the amplification step by documenting the NTM of two genomic segments of FBNSV. We argue that the NTM must be documented and carefully considered when determining the sensitivity and accuracy of data from NGS studies.

IMPORTANCE The advent of next-generation sequencing (NGS) techniques now enables study of the genetic diversity of viral populations. A good understanding of the genetic structure of these populations first requires the ability to identify the different isolates or variants and second requires the ability to accurately quantify them. Prior to sequencing, viral genomes need to be amplified, a step that potentially imposes quantitative biases and modifies the viral population structure. In particular, the number of target molecules (NTM) used during the amplification step is of primary importance, as it sets the limit of the accuracy and sensitivity of the sequencing procedure. In this work, we used 20 replicated populations of the multipartite faba bean necrotic stunt virus (FBNSV) to estimate the various limitations of ultradeep-sequencing studies performed on intrahost viral populations. We report quantitative biases during rolling-circle amplification and the NTM of two genomic segments of FBNSV.

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The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies
Romain Gallet, Frédéric Fabre, Yannis Michalakis, Stéphane Blanc
Journal of Virology Jul 2017, 91 (16) e00561-17; DOI: 10.1128/JVI.00561-17

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The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies
Romain Gallet, Frédéric Fabre, Yannis Michalakis, Stéphane Blanc
Journal of Virology Jul 2017, 91 (16) e00561-17; DOI: 10.1128/JVI.00561-17
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KEYWORDS

DNA sequencing
FBNSV
faba bean
Medicago truncatula
next-generation sequencing
number of target molecules
rolling-circle amplification
sequencing accuracy
sequencing sensitivity

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