Optimized library preparation, sequencing, and data analysis protocols for the generation of orbivirus consensus sequences

Tillie J Dunham; Tyler J Sherman; Kirsten J Reed; Corey Brelsfoard; Tavis K Anderson; Lee W Cohnstaedt; Mark D Stenglein; Christie E Mayo

doi:10.1186/s12864-025-12422-y

Optimized library preparation, sequencing, and data analysis protocols for the generation of orbivirus consensus sequences

BMC Genomics. 2026 Jan 13;27(1):48. doi: 10.1186/s12864-025-12422-y.

Authors

Tillie J Dunham¹, Tyler J Sherman², Kirsten J Reed², Corey Brelsfoard³, Tavis K Anderson⁴, Lee W Cohnstaedt⁵, Mark D Stenglein¹, Christie E Mayo⁶

Affiliations

¹ Center for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
² Diagnostic Medicine Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
³ Department of Biological Sciences, Texas Tech University, Lubbock, TX, USA.
⁴ National Animal Disease Center, Agricultural Research Service, Department of Agriculture, Ames, IA, USA.
⁵ Agricultural Research Service, Department of Agriculture, National Bio and Agro-Defense Facility, Manhattan, KS, USA.
⁶ Center for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA. christie.mayo@colostate.edu.

Abstract

Whole genome sequencing (WGS) via next-generation sequencing (NGS) platforms offers a powerful approach for investigating viral genetic diversity. Orbiviruses are an economically important group of arboviruses with a double-stranded RNA (dsRNA) genome consisting of 10 segments. Traditional approaches to virus sequencing relied primarily on processing with various treatments such as lithium chloride to enrich dsRNAs and then sequencing on Illumina platforms due to its lower error rate. To reduce the time and cost associated with orbivirus sequencing, we simplified the sample preparation, sequencing, and analysis protocols. Our optimized sample and library preparation protocols achieved comparable results to established methods while benefiting from simpler sample preparation. The optimized protocols for Illumina and Nanopore platforms (which produces longer reads) resulted in both platforms producing high quality whole genome sequences. To streamline the data analysis, we developed OrbiSeq, a reproducible Nextflow workflow for analysis and consensus sequence generation of orbivirus genomes from Illumina and Nanopore sequence data. While the OrbiSeq pipeline was optimized with orbivirus sequences, it can be used to recover consensus sequences for any segmented or non-segmented viral genome, provided that the sequence is sufficiently similar to existing reference sequences. The optimized protocols for producing viral sequences enhance opportunities for extensive genomic surveillance and in turn deeper evolutionary insights.

Keywords: Library preparation; Next-Generation sequencing (NGS); Nextflow; OrbiSeq pipeline; Orbivirus; Reproducibility; Sample preparation; Segmented genome; Viral genomics; Whole genome sequencing (WGS).

MeSH terms

Consensus Sequence*
Data Analysis
Gene Library*
Genome, Viral
High-Throughput Nucleotide Sequencing* / methods
Orbivirus* / genetics
Whole Genome Sequencing* / methods