Dataset Information

Genomic analysis of human noroviruses using combined Illumina-Nanopore data.

ABSTRACT: Whole-genome sequence analysis of noroviruses is routinely performed by employing a metagenomic approach. While this methodology has several advantages, such as allowing for the examination of co-infection, it has some limitations, such as the requirement of high viral load to achieve full-length or near full-length genomic sequences. In this study, we used a pre-amplification step to obtain full-length genomic amplicons from 39 Canadian GII isolates, followed by deep sequencing on Illumina and Oxford Nanopore platforms. This approach significantly reduced the required viral titre to obtain full-genome coverage. Herein, we compared the coverage and sequences obtained by both platforms and provided an in-depth genomic analysis of the obtained sequences, including the presence of single-nucleotide variants and recombination events.

SUBMITTER: Flint A

PROVIDER: S-EPMC8570145 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Genomic analysis of human noroviruses using combined Illumina-Nanopore data.

Flint Annika A Reaume Spencer S Harlow Jennifer J Hoover Emily E Weedmark Kelly K Nasheri Neda N

Virus evolution 20210915 2

Whole-genome sequence analysis of noroviruses is routinely performed by employing a metagenomic approach. While this methodology has several advantages, such as allowing for the examination of co-infection, it has some limitations, such as the requirement of high viral load to achieve full-length or near full-length genomic sequences. In this study, we used a pre-amplification step to obtain full-length genomic amplicons from 39 Canadian GII isolates, followed by deep sequencing on Illumina and ...[more]

PMID: 35186325

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Project description:BackgroundWe benchmarked the hybrid assembly approaches of MaSuRCA, SPAdes, and Unicycler for bacterial pathogens using Illumina and Oxford Nanopore sequencing by determining genome completeness and accuracy, antimicrobial resistance (AMR), virulence potential, multilocus sequence typing (MLST), phylogeny, and pan genome. Ten bacterial species (10 strains) were tested for simulated reads of both mediocre- and low-quality, whereas 11 bacterial species (12 strains) were tested for real reads.ResultsUnicycler performed the best for achieving contiguous genomes, closely followed by MaSuRCA, while all SPAdes assemblies were incomplete. MaSuRCA was less tolerant of low-quality long reads than SPAdes and Unicycler. The hybrid assemblies of five antimicrobial-resistant strains with simulated reads provided consistent AMR genotypes with the reference genomes. The MaSuRCA assembly of Staphylococcus aureus with real reads contained msr(A) and tet(K), while the reference genome and SPAdes and Unicycler assemblies harbored blaZ. The AMR genotypes of the reference genomes and hybrid assemblies were consistent for the other five antimicrobial-resistant strains with real reads. The numbers of virulence genes in all hybrid assemblies were similar to those of the reference genomes, irrespective of simulated or real reads. Only one exception existed that the reference genome and hybrid assemblies of Pseudomonas aeruginosa with mediocre-quality long reads carried 241 virulence genes, whereas 184 virulence genes were identified in the hybrid assemblies of low-quality long reads. The MaSuRCA assemblies of Escherichia coli O157:H7 and Salmonella Typhimurium with mediocre-quality long reads contained 126 and 118 virulence genes, respectively, while 110 and 107 virulence genes were detected in their MaSuRCA assemblies of low-quality long reads, respectively. All approaches performed well in our MLST and phylogenetic analyses. The pan genomes of the hybrid assemblies of S. Typhimurium with mediocre-quality long reads were similar to that of the reference genome, while SPAdes and Unicycler were more tolerant of low-quality long reads than MaSuRCA for the pan-genome analysis. All approaches functioned well in the pan-genome analysis of Campylobacter jejuni with real reads.ConclusionsOur research demonstrates the hybrid assembly pipeline of Unicycler as a superior approach for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.

Project description:Genomic surveillance enables the early detection of pathogen transmission in healthcare facilities and contributes to the reduction of substantial patient harm. Fast turnaround times, flexible multiplexing, and low capital requirements make Nanopore sequencing well suited for genomic surveillance purposes; the analysis of Nanopore data, however, can be challenging. We present NanoCore, a user-friendly method for Nanopore-based genomic surveillance in healthcare facilities, enabling the calculation and visualization of cgMLST-like (core-genome multilocus sequence typing) sample distances directly from unassembled Nanopore reads. NanoCore implements a mapping, variant calling, and multilevel filtering strategy and also supports the analysis of Illumina data. We validated NanoCore on two 24-isolate data sets of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). In the Nanopore-only mode, NanoCore-based pairwise distances between closely related isolates were near-identical to Illumina-based SeqSphere+ distances, a gold standard commercial method (average differences of 0.75 and 0.81 alleles for MRSA and VRE; sd = 0.98 and 1.00), and gave an identical clustering into closely related and non-closely related isolates. In the "hybrid" mode, in which only Nanopore data are used for some isolates and only Illumina data for others, increased average pairwise isolate distance differences were observed (average differences of 3.44 and 1.95 for MRSA and VRE, respectively; sd = 2.76 and 1.34), while clustering results remained identical. NanoCore is computationally efficient (<15 hours of wall time for the analysis of a 24-isolate data set on a workstation), available as free software, and supports installation via conda. In conclusion, NanoCore enables the effective use of the Nanopore technology for bacterial pathogen surveillance in healthcare facilities.ImportanceGenomic surveillance involves sequencing the genomes and measuring the relatedness of bacteria from different patients or locations in the same healthcare facility, enabling an improved understanding of pathogen transmission pathways and the detection of "silent" outbreaks that would otherwise go undetected. It has become an indispensable tool for the detection and prevention of healthcare-associated infections and is routinely applied by many healthcare institutions. The earlier an outbreak or transmission chain is detected, the better; in this context, the Oxford Nanopore sequencing technology has important potential advantages over traditionally used short-read sequencing technologies, because it supports "real-time" data generation and the cost-effective "on demand" sequencing of small numbers of bacterial isolates. The analysis of Nanopore sequencing data, however, can be challenging. We present NanoCore, a user-friendly software for genomic surveillance that works directly based on Nanopore sequencing reads in FASTQ format, and demonstrate that its accuracy is equivalent to traditional gold standard short read-based analyses.

Project description:BackgroundStructural variants (SVs), including deletions, insertions, duplications, and inversions, are relatively long genomic variations implicated in a diverse range of processes from human disease to ecology and evolution. Given their complex signatures, tendency to occur in repeated regions, and large size, discovering SVs based on short reads is challenging compared to single-nucleotide variants. The increasing availability of long-read technologies has greatly facilitated SV discovery; however, these technologies remain too costly to apply routinely to population-level studies. Here, we combined short-read and long-read sequencing technologies to provide a comprehensive population-scale assessment of structural variation in a panel of Canadian soybean cultivars.ResultsWe used Oxford Nanopore long-read sequencing data (~12× mean coverage) for 17 samples to both benchmark SV calls made from Illumina short-read data and predict SVs that were subsequently genotyped in a population of 102 samples using Illumina data. Benchmarking results show that variants discovered using Oxford Nanopore can be accurately genotyped from the Illumina data. We first use the genotyped deletions and insertions for population genetics analyses and show that results are comparable to those based on single-nucleotide variants. We observe that the population frequency and distribution within the genome of deletions and insertions are constrained by the location of genes. Gene Ontology and PFAM domain enrichment analyses also confirm previous reports that genes harboring high-frequency deletions and insertions are enriched for functions in defense response. Finally, we discover polymorphic transposable elements from the deletions and insertions and report evidence of the recent activity of a Stowaway MITE.ConclusionsWe show that structural variants discovered using Oxford Nanopore data can be genotyped with high accuracy from Illumina data. Our results demonstrate that long-read and short-read sequencing technologies can be efficiently combined to enhance SV analysis in large populations, providing a reusable framework for their study in a wider range of samples and non-model species.

Dataset Information

Genomic analysis of human noroviruses using combined Illumina-Nanopore data.

Publications

Genomic analysis of human noroviruses using combined Illumina-Nanopore data.

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