Abstract
BackgroundNext-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools.MethodsIn this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform.ResultsTwenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25–30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2–3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample.ConclusionsThis work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples.
Highlights
Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids
Efficient protocol for multiplexed sequencing using a single flow cell on the Illumina MiSeq platform coupled with a detailed customized Galaxy workflow for de novo assembly that allows for quick and accurate generation of near-full-length, or full-length, genome sequences of dozens of isolates, simultaneously
Nucleic acids quantification and libraries fragment size The nucleic acid concentrations obtained at different steps throughout the preparation of the libraries for sequencing are summarized in Additional file 3: Table S2
Summary
Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. Conventional laboratory methods like enzyme-linked immunosorbent assay, nucleic acid hybridization technique, and polymerase chain reaction (PCR) are all common and inexpensive diagnostic and research tools utilized in virology [1]. As these assays are highly dependent on reagents (primers, probes, antibodies) developed from previously known and characterized viruses, they might be ineffective for the identification of new viral variants, new pathogens or a mixed population of pathogens that have high genetic divergence from those described previously [1, 2]. This has led to the discovery of numerous viral pathogens [4,5,6], including 2009 pandemic influenza A, a novel pegivirus, Canine bocavirus 3, and a novel hepacivirus [7,8,9,10]
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