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01.12.2017 | Methodology | Ausgabe 1/2017 Open Access

Virology Journal 1/2017

A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses

Zeitschrift:
Virology Journal > Ausgabe 1/2017
Autoren:
Kiril M. Dimitrov, Poonam Sharma, Jeremy D. Volkening, Iryna V. Goraichuk, Abdul Wajid, Shafqat Fatima Rehmani, Asma Basharat, Ismaila Shittu, Tony M. Joannis, Patti J. Miller, Claudio L. Afonso
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12985-017-0741-5) contains supplementary material, which is available to authorized users.

Abstract

Background

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. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools.

Methods

In 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.

Results

Twenty-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.

Conclusions

This 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.
Zusatzmaterial
Additional file 1: Table S1. Background information of the avian paramyxovirus isolates used in this study. (DOCX 15 kb)
12985_2017_741_MOESM1_ESM.docx
Additional file 2: Figure S1. Major processing steps used in the current study (PDF 222 kb)
12985_2017_741_MOESM2_ESM.pdf
Additional file 3: Table S2. Nucleic acid concentrations and library fragment size distributions of thirty virus isolates used in the study. (DOCX 18 kb)
12985_2017_741_MOESM3_ESM.docx
Additional file 4: Table S3. Sequences of primers used for sequencing internal gaps and missing termini. (DOCX 13 kb)
12985_2017_741_MOESM4_ESM.docx
Additional file 5: Table S4. Time and cost analysis of next-generation sequencing of thirty avian paramyxovirus isolates. (DOCX 15 kb)
12985_2017_741_MOESM5_ESM.docx
Additional file 6: Figure S2. Phylogenetic analysis based on the complete genome coding sequence of Newcastle disease virus isolates studied here and selected closely related sequences from GenBank. Figure S3. Phylogenetic analysis based on the hypervariable region of the spike protein gene of Infectious bronchitis virus studied here and selected closely related sequences from GenBank. (PDF 142 kb)
12985_2017_741_MOESM6_ESM.pdf
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