An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

AbstractHow viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluated our multiplexed amplicon approach - PrimalSeq - to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We developed an experimental protocol and computational tool (iVar) for using PrimalSeq to measure virus diversity using Illumina and compared the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.

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Genetic variation in West Nile virus from naturally infected mosquitoes and birds suggests quasispecies structure and strong purifying selection

Journal of General Virology ◽

10.1099/vir.0.81015-0 ◽

2005 ◽

Vol 86 (8) ◽

pp. 2175-2183 ◽

Cited By ~ 137

Author(s):

Greta Jerzak ◽

Kristen A. Bernard ◽

Laura D. Kramer ◽

Gregory D. Ebel

Keyword(s):

Genetic Diversity ◽

West Nile Virus ◽

Consensus Sequence ◽

Purifying Selection ◽

Infected Mosquito ◽

Structural Protein ◽

Coding Region ◽

West Nile ◽

Transmission Cycle ◽

The Mean

Intrahost genetic diversity was analysed in naturally infected mosquitoes and birds to determine whether West Nile virus (WNV) exists in nature as a quasispecies and to quantify selective pressures within and between hosts. WNV was sampled from ten infected birds and ten infected mosquito pools collected on Long Island, NY, USA, during the peak of the 2003 WNV transmission season. A 1938 nt fragment comprising the 3′ 1159 nt of the WNV envelope (E) coding region and the 5′ 779 nt of the non-structural protein 1 (NS1) coding region was amplified and cloned and 20 clones per specimen were sequenced. Results from this analysis demonstrate that WNV infections are derived from a genetically diverse population of genomes in nature. The mean nucleotide diversity was 0·016 % within individual specimens and the mean percentage of clones that differed from the consensus sequence was 19·5 %. WNV sequences in mosquitoes were significantly more genetically diverse than WNV in birds. No host-dependent bias for particular types of mutations was observed and estimates of genetic diversity did not differ significantly between E and NS1 coding sequences. Non-consensus clones obtained from two avian specimens had highly similar genetic signatures, providing preliminary evidence that WNV genetic diversity may be maintained throughout the enzootic transmission cycle, rather than arising independently during each infection. Evidence of purifying selection was obtained from both intra- and interhost WNV populations. Combined, these data support the observation that WNV populations may be structured as a quasispecies and document strong purifying natural selection in WNV populations.

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Genetic Diversity in the Collaborative Cross Model Recapitulates Human West Nile Virus Disease Outcomes

mBio ◽

10.1128/mbio.00493-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 55

Author(s):

Jessica B. Graham ◽

Sunil Thomas ◽

Jessica Swarts ◽

Aimee A. McMillan ◽

Martin T. Ferris ◽

...

Keyword(s):

Genetic Diversity ◽

West Nile Virus ◽

Virus Infection ◽

Immune Responses ◽

West Nile Virus Infection ◽

Collaborative Cross ◽

Adaptive Immune Responses ◽

West Nile ◽

Adaptive Immune ◽

Wide Range

ABSTRACT West Nile virus (WNV) is an emerging neuroinvasive flavivirus that now causes significant morbidity and mortality worldwide. The innate and adaptive immune responses to WNV infection have been well studied in C57BL/6J inbred mice, but this model lacks the variations in susceptibility, immunity, and outcome to WNV infection that are observed in humans, thus limiting its usefulness to understand the mechanisms of WNV infection and immunity dynamics. To build a model of WNV infection that captures human infection outcomes, we have used the Collaborative Cross (CC) mouse model. We show that this model, which recapitulates the genetic diversity of the human population, demonstrates diversity in susceptibility and outcomes of WNV infection observed in humans. Using multiple F1 crosses of CC mice, we identified a wide range of susceptibilities to infection, as demonstrated through differences in survival, clinical disease score, viral titer, and innate and adaptive immune responses in both peripheral tissues and the central nervous system. Additionally, we examined the Oas1b alleles in the CC mice and confirmed the previous finding that Oas1b plays a role in susceptibility to WNV; however, even within a given Oas1b allele status, we identified a wide range of strain-specific WNV-associated phenotypes. These results confirmed that the CC model is effective for identifying a repertoire of host genes involved in WNV resistance and susceptibility. The CC effectively models a wide range of WNV clinical, virologic, and immune phenotypes, thus overcoming the limitations of the traditional C57BL/6J model, allowing genetic and mechanistic studies of WNV infection and immunity in differently susceptible populations. IMPORTANCE Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans.

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Exploring Quantitative Metagenomics Studies using Oxford Nanopore Sequencing: A Computational and Experimental Protocol

10.21203/rs.3.rs-131495/v1 ◽

2020 ◽

Author(s):

Rohia ALILI ◽

Eugeni BELDA ◽

Karine CLEMENT ◽

Phuong Le ◽

Edi PRIFTI ◽

...

Keyword(s):

Dna Extraction ◽

Gut Microbiome ◽

Large Scale ◽

Low Cost ◽

Read Length ◽

Whole Genome ◽

Nanopore Sequencing ◽

Experimental Protocol ◽

Microbial Composition ◽

Oxford Nanopore

Abstract Background: The gut microbiome plays a major role in chronic diseases, several of which are characterized by an altered diversity and composition of bacterial communities. Large-scale sequencing projects allowed the characterization of these microbial community perturbations. However, a gap remains in how these discoveries can be translated into clinical applications. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed.Results: Here, we propose a computational and experimental protocol for whole genome quantitative metagenomics studies of the human gut microbiome with Oxford Nanopore sequencing technology (ONT). We developed a bioinformatic pipeline to process ONT sequences based on the evaluation of different alignment parameters in the estimation of microbial diversity and composition. We also optimized stool collection and DNA extraction methods to maximize read length, a critical parameter for the sequence alignment and classification. Our analytical pipeline was evaluated using simulations of metagenomic communities to reflect naturally occuring compositional variations. We then validated our experimental and analytical pipeline with stool samples from a bariatric surgery cohort sequenced with ONT and Illumina, revealing comparable diversity and microbial composition profiles. These results were compared to those previously obtained with SOLiD sequencing, where differences were observed, possibly explained by variations in library preparation steps. Finally, we found that sequences obtained with ONT allowed assembly of complete genomes for disease-related species.Conclusion: This protocol can be implemented in the clinical or individual setting, bringing rapid personalized whole genome profiling of target microbiome species. Keywords: quantitative metagenomics, microbiome, obesity, gut microbiota, microbial DNA extraction, sequencing, Simulation, Oxford Nanopore Technologies, MinION.

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West Nile Virus in Brazil

Pathogens ◽

10.3390/pathogens10070896 ◽

2021 ◽

Vol 10 (7) ◽

pp. 896

Author(s):

Érica Azevedo Costa ◽

Marta Giovanetti ◽

Lilian Silva Catenacci ◽

Vagner Fonseca ◽

Flávia Figueira Aburjaile ◽

...

Keyword(s):

West Nile Virus ◽

Theoretical Perspective ◽

Phylogenetic Inference ◽

Genomic Diversity ◽

Sufficient Evidence ◽

Nanopore Sequencing ◽

Serological Evidence ◽

West Nile ◽

Epidemiological Modeling ◽

Diagnostic Assays

Background: West Nile virus (WNV) was first sequenced in Brazil in 2019, when it was isolated from a horse in the Espírito Santo state. Despite multiple studies reporting serological evidence suggestive of past circulation since 2004, WNV remains a low priority for surveillance and public health, such that much is still unknown about its genomic diversity, evolution, and transmission in the country. Methods: A combination of diagnostic assays, nanopore sequencing, phylogenetic inference, and epidemiological modeling are here used to provide a holistic overview of what is known about WNV in Brazil. Results: We report new genetic evidence of WNV circulation in southern (Minas Gerais, São Paulo) and northeastern (Piauí) states isolated from equine red blood cells. A novel, climate-informed theoretical perspective of the potential transmission of WNV across the country highlights the state of Piauí as particularly relevant for WNV epidemiology in Brazil, although it does not reject possible circulation in other states. Conclusion: Our output demonstrates the scarceness of existing data, and that although there is sufficient evidence for the circulation and persistence of the virus, much is still unknown on its local evolution, epidemiology, and activity. We advocate for a shift to active surveillance, to ensure adequate preparedness for future epidemics with spill-over potential to humans.

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ONTdeCIPHER: An amplicon-based nanopore sequencing pipeline for tracking pathogen variants

10.1101/2021.10.13.464242 ◽

2021 ◽

Author(s):

Emira Cherif ◽

Fatou Seck Thiam ◽

Mohammad Salma ◽

Georgina RIVERA-INGRAHAM ◽

Fabienne Justy ◽

...

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analyses ◽

Command Line ◽

Nanopore Sequencing ◽

Viral Isolate ◽

Sequence Alignments ◽

Molecular Surveillance ◽

Oxford Nanopore ◽

Quality Testing ◽

Downstream Analysis

Motivation: Amplicon-based nanopore sequencing is increasingly used for molecular surveillance during epidemics (e.g. ZIKA, EBOLA) or pandemics (e.g. SARS-CoV-2). However, there is still a lack of versatile and easy-to-use tools that allow users with minimal bioinformatics skills to perform the main steps of downstream analysis, from quality testing to SNPs effect to phylogenetic analysis. Results: Here, we present ONTdeCIPHER, an amplicon-based Oxford Nanopore Technology (ONT) sequencing pipeline to analyze the genetic diversity of SARS-CoV-2 and other pathogenes. Our pipeline integrates 13 bioinformatics tools. With a single command line and a simple configuration file, users can pre-process their data and obtain the sequencing statistics, reconstruct the consensus genome, identify variants and their effects for each viral isolate, infer lineage and, finally perform multi-sequence alignments and phylogenetic analyses.

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