Comparable outcomes from long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory samples

Authors: BUTT, SALMAN - Cornell University; KARIITHI, HENRY - Orise Fellow; VOLKENING, JEREMY - Base2bio; Taylor, Tonya; LEYSON, CHRISTINA - Orise Fellow; Pantin Jackwood, Mary; Suarez, David; STANTON, JAMES - University Of Georgia; Afonso, Claudio

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2022
Publication Date: 12/1/2022
Citation: Butt, S.L., Kariithi, H.M., Volkening, J.D., Taylor, T.L., Leyson, C., Pantin Jackwood, M.J., Suarez, D.L., Stanton, J.B., Afonso, C.L. 2022. Comparable outcomes from long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory samples. Frontiers in Veterinary Science. 9:1073919. https://doi.org/10.3389/fvets.2022.1073919.
DOI: https://doi.org/10.3389/fvets.2022.1073919

Interpretive Summary: Respiratory diseases are a significant threat to the global poultry industry. Co-infections with different microbial pathogens produce respiratory disease complexes and complicate the accurate disease diagnosis when using target-specific tests. Next generation sequencing (NGS) provides the possibility for simultaneous diagnostic testing and sequencing of many pathogens directly from a clinical sample. In the current study, a multiplexed, random sequencing approach based on MinION nanopore sequencing was developed and compared to other pathogen detection methods (RT-qPCR and Illumina MiSeq) for the detection of viral and bacterial co-infections in commercial poultry. Two different types of sample were sequenced in this study: experimental samples from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS), and field-collected clinical samples from chickens from live bird markets. The MinION sequencing approach provided a faster and accurate characterization of the co-infecting viral and bacterial pathogens in the samples examined.

Technical Abstract: Co-infections of avian species with different RNA viruses and pathogenic bacteria are often misdiagnosed or incompletely characterized using targeted diagnostic methods, which could affect the accurate management of clinical disease. A non-targeted sequencing approach with rapid and precise characterization of pathogens should help respiratory disease management by providing a comprehensive view of the causes of disease. Long-read portable sequencers have significant potential advantages over established short-read sequencers due to portability, speed, and lower cost. The applicability of short reads random sequencing for direct detection of pathogens in clinical poultry samples has been previously demonstrated. Here we demonstrate the feasibility of long read random sequencing approaches to identify disease agents in clinical samples. Experimental oropharyngeal swab samples (n = 12) from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS) and field-collected clinical oropharyngeal swab samples (n = 11) from Kenyan live bird markets previously testing positive for Newcastle disease virus (NDV) were randomly sequenced on the MinION platform and results validated by comparing to real time PCR and short read random sequencing in the Illumina MiSeq platform. In the swabs from experimental infections, each of three agents in every RT-qPCR-positive sample (Ct range 19–34) was detectable within 1 h on the MinION platform, except for AIV one agent in one sample (Ct = 36.21). Nine of 12 IBV-positive samples were assigned genotypes within 1 h, as were five of 11 AIV-positive samples. MinION relative abundances of the test agent (AIV, IBV and MS) were highly correlated with RT-qPCR Ct values (R range-0.82 to-0.98). In field-collected clinical swab samples, NDV (Ct range 12–37) was detected in all eleven samples within 1 h of MinION sequencing, with 10 of 11 samples accurately genotyped within 1 h. All NDV-positive field samples were found to be co-infected with one or more additional respiratory agents. These results demonstrate that MinION sequencing can provide rapid, and sensitive non-targeted detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the Illumina MiSeq.