Ampliseq for Illumina Technology Enables Detailed Molecular Epidemiology of Rabies Lyssaviruses from Infected Formalin-Fixed Paraffin-Embedded Tissues.

Whole genome sequencing of rabies lyssaviruses (RABVs) has enabled the generation of highly detailed phylogenies that reveal viral transmission patterns of disease in reservoir species. Such information is highly important for informing best practices with respect to wildlife rabies control. However, specimens available only as formalin fixed paraffin embedded (FFPE) samples have been recalcitrant to such analyses. Due to the damage inflicted by tissue processing, only relatively short amplicons can be generated by standard RT-PCR methods, making the generation of full-length genome sequences very tedious. While highly parallel shotgun sequencing of total RNA can potentially overcome these challenges, the low percentage of reads representative of the virus may be limiting. Ampliseq technology enables massively multiplex amplification of nucleic acids to produce large numbers of short PCR products. Such a strategy has been applied to the sequencing of entire viral genomes but its use for rabies virus analysis has not been reported previously. This study describes the generation of an Ampliseq for Illumina primer panel, which was designed based on the global sequence diversity of rabies viruses, and which enables efficient viral genome amplification and sequencing of rabies-positive FFPE samples. The subsequent use of such data for detailed phylogenetic analysis of the virus is demonstrated.

Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis.

The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.