Long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory clinical swabs

Authors: BUTT, SALMAN - University Of Georgia; KARITHI, HENRY - Orise Fellow; Taylor, Tonya; VOLKENING, JEREMY - Base2bio; LEYSON, CHRISTINA - Orise Fellow; Pantin Jackwood, Mary; JACKWOOD, MARK - University Of Georgia; FERGUSON, NAOLA - University Of Georgia; Suarez, David; Afonso, Claudio

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 10/30/2020
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Objectives: Co-infections of poultry with different RNA viruses and bacteria are often misdiagnosed and affect the accurate diagnosis of clinical disease. A metagenomic approach with rapid and precise characterization of these pathogens is crucial for respiratory disease management. The ability of long and short reads sequencing approaches to rapidly detect viral and bacterial pathogens from clinical samples was evaluated. Methods: Three different type of sample sets; 1) experimental oral swab samples (n = 12) with Infectious Bronchitis Virus (IBV), Avian Influenza Virus (AIV) and Mycoplasma synoviae (MS) ; field collected clinical oral swab samples (n = 12) from commercial poultry in Pakistan and 3) field collected clinical oral swab samples (n = 12) from live bird markets in Kenya were sequenced with MinION-metagenomic approach. Briefly, total RNA was extracted, randomly reverse transcribed, barcoded double-stranded cDNA libraries and pooled (n = 12) for sequencing. These samples were also sequenced on MiSeq for comparison sake. For quantitative detection of pathogens in these samples, read based analyses of MinION and MiSeq data were performed using BLAST on customized databases. Results: In each of the samples sets, microbial reads were detected within 30 minutes of MinION sequencing and accurately characterized for their genetic types. In experimental swabs, the MinION read counts for each of the pathogen (AIV, IBV and MS) were comparable with the respective RT-qPCR Ct values in all samples (12/12). In field collected samples, MinION and MiSeq approaches detected NDV in Kenya samples (12/12) and in Pakistani samples (12/12). Bioinformatics analysis of MinION and MiSeq data lead to the assembly of multiple NDV complete genomes (genotype Vd) from Kenya samples and multiple NDV (genotype VIIi) and AIV (H9N2,) genomes from Pakistani samples. Conclusions: Together, these results suggest that MinION provides a rapid, multiplexed and cost-effective detection of respiratory pathogens in clinical samples and pathogen identification is comparable to MiSeq platform.