The number of target molecules of the amplification step limits accuracy and sensitivity in ultradeep-sequencing viral population studies

BGPI : équipe 2

إنجليزي. The invention of next generation sequencing techniques (NGS) marked the coming of a new era in the detection of genetic diversity of intra-host viral populations. A good understanding of the genetic structure of these populations first requires being able to identify the different isolates or variants, and second 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, though of primary importance as it sets the limit of the accuracy and the sensitivity of the sequencing procedure. In the present article, we investigated quantitative biases in the NGS study of populations of a multipartite ssDNA virus at different steps of the procedure. We studied 20 independent populations of the ssDNA Faba Bean Necrotic Stunt Virus (FBNSV) virus in two host plants, Faba bean and Medicago. The 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 significant distortion of the FBNSV genome formula after the amplification and the sequencing steps. We also quantified the genetic bottleneck occurring at the amplification step by documenting the NTM of two genomic segments of the FBNSV. We argue that the NTM must be documented and carefully considered when interpreting the sensitivity and accuracy of NGS studies.Importance The advent of next generation sequencing techniques (NGS) now enables studying the genetic diversity of viral populations. A good understanding of the genetic structure of these populations first requires being able to identify the different isolates or variants, and second 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 the 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 ultra deep sequencing studies performed on intra-host viral populations. We report quantitative biases during Rolling Circle Amplification and the NTM of two genomic segments of the FBNSV.