The 28S rRNA RT-qPCR assay for host depletion evaluation to enhance avian virus detection in illumina and nanopore sequencing

Authors: GORAICHUK, IRYNA - Oak Ridge Institute For Science And Education (ORISE); HARDEN, MARK - Tuskegee University; Spackman, Erica; Suarez, David

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/9/2024
Publication Date: 1/31/2023
Citation: Goraichuk, I.V., Harden, M., Spackman, E., Suarez, D.L. 2023. The 28S rRNA RT-qPCR assay for host depletion evaluation to enhance avian virus detection in illumina and nanopore sequencing. Frontiers in Microbiology. Volume 15. https://doi.org/10.3389/fmicb.2024.1328987.
DOI: https://doi.org/10.3389/fmicb.2024.1328987

Interpretive Summary: The use of Next Generation Sequencing (NGS) techniques continue to identify pathogens in clinical samples continues to improve. Often full length viral genomes can be identified in samples and this sequence information can be used to not only identify what pathogens are in samples, but also determine the genotype, pathotype, or lineage of a virus. Using current NGS methods, a large number of sequence reads are actually host or bacterial reads. We have previously published a method to deplete host RNA with the goal of having more viral reads in each sample. In an effort to improve on this earlier method, we have tested several alternative methods, and the method that improved the results the best was to use a new enzyme to remove more DNA from the sample. The residual host DNA was reducing the sequence results. Using this new method, we anticipate more viral sequences per sample will be produced and that will improve the overall sequence results.

Technical Abstract: Abundant host and bacterial sequences can obscure the detection of less prevalent viruses in un-targeted next-generation sequencing (NGS). Efficient removal of these non-targeted sequences is vital for accurate viral detection. This study presents a novel 28S ribosomal RNA (rRNA) RT-qPCR assay designed to assess the efficiency of avian rRNA depletion before conducting costly NGS for the detection of avian RNA viruses. The comprehensive evaluation of this 28S-test focuses on substituting DNase I with the alternative DNase in our established depletion protocols and finetuning essential parameters for reliable host rRNA depletion. To validate the effectiveness of the 28S-test, we compared its performance with NGS results obtained from both Illumina and Nanopore sequencing platforms. This evaluation utilized swab samples from chickens infected with highly pathogenic avian influenza virus, subjected to established and modified depletion protocols. Both methods significantly reduced host rRNA levels, but using the alternative DNase had superior performance. Additionally, utilizing the 28S-test, we explored cost- and time-effective strategies, such as reduced probe concentrations and other alternative DNase usage, assessed the impact of filtration pre-treatment, and evaluated various experimental parameters to further optimize the depletion protocol. Our findings underscore the value of the 28S-test in optimizing depletion methods for advancing improvements in avian disease research through NGS.