A beginner’s guide for FMDV quasispecies analysis: sub-consensus variant detection and haplotype reconstruction using next-generation sequencing

Abstract

Deep sequencing of viral genomes is a powerful tool to study RNA virus complexity. However, the analysis of next-generation sequencing data might be challenging for researchers who have never approached the study of viral quasispecies by this methodology. In this work we present a suitable and affordable guide to explore the sub-consensus variability and to reconstruct viral quasispecies from Illumina sequencing data. The guide includes a complete analysis pipeline along with user-friendly descriptions of software and file formats. In addition, we assessed the feasibility of the workf low proposed by analyzing a set of foot-and-mouth disease viruses (FMDV) with different degrees of variability. This guide introduces the analysis of quasispecies of FMDV and other viruses through this kind of approach.

Testing the genomic stability of the Brazilian yellow fever vaccine strain using next-generation sequencing data

“…The V2IDA pipeline was used to analyse the 17DD strain genetic diversity. To choose the algorithms enrolled in the V2IDA pipeline, nine state-of-the-art algorithms (three for each step) were previously selected after careful literature revision (electronic supplementary material, table S4) from previous experiments [ 19 , 22 , 23 , 32 , 33 , 35 , 36 , 38 , 39 ]. The chosen algorithms have the same fundamental characteristics: open-source, executable from the command line and widely used in the scientific community (including in studies with viral RNA genomes).…”

“…Highlighting the importance of our study, we reinforce the monitoring of genetic diversity and in silico genetic stability testing as part of the vaccine manufacturing process to ensure the safety of all vaccine lots administered to the population. However, existing pipelines that analyse viral NGS samples do not accurately extract genetic diversity royalsocietypublishing.org/journal/rsfs Interface Focus 11: 20200063 information when dealing with viral vaccine samples due to the lack of specific parameters [22,38], use of inappropriate tools [19,23,36], and not performing quasispecies reconstruction [15,35,37,38]. This often leads to false results and affects negatively the sensitivity and specificity metrics.…”

A paucigranulocytic asthma host environment promotes the emergence of virulent influenza viral variants

“…The haplotypes for each sample were reconstructed for each gene segment using a previously published pipeline ( Cacciabue et al, 2020 ). In brief, FastQC ( Andrews, 2010 ) was used for quality assurance of the NGS paired-end raw reads followed by BBtools ( Bushnell, 2014 ), for removing and filtering adapters and low-quality reads.…”

QuasiFlow: a bioinformatic tool for genetic variability analysis from next generation sequencing data

“…Fortunately, the advent of next-generation sequencing (NGS) technologies enabled the fast and cheap detection of any minor variant within a huge population of genomes (Bull et al 2016; Posada-Cespedes et al 2017) and discern all single nucleotide variations (SNVs) in any quasispecies to define a haplotype (viral variant) without the need of culture or cloning prior to sequencing (Zukurov et al 2016). However, analysing NGS data can be an awkward endeavour starting from the available open-source, cutting-edge software with incompatible formats to study recombination, variant calling and haplotypes (Cacciabue et al 2020). Additionally, quasispecies studies must deal with sequencing errors, whose frequency lays close to the viral replication error rates (10 -3 -10 -5 errors per nucleotide).…”