Performance evaluation of host depletion methods for improved detection of avian viruses via Illumina and Nanopore sequencing

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

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/6/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary:

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 rRNAs is vital for accurate viral detection. In this study, we conducted an evaluation of the host depletion methods aimed at enhancing the detection of avian viruses through NGS. Our investigation primarily focused on the substitution of DNase I with TURBO DNase within our established RNase H-based depletion protocols to reduce the abundance of host and bacterial reads using DNA probes designed to target chicken 18S and 28S rRNA, specific chicken mitochondrial RNA, and select 16S and 23S bacterial rRNA. We validated the performance of the modified depletion protocol by comparing the NGS results obtained using both Illumina and Nanopore sequencing platforms. To carry out this evaluation, we utilized clinical swabs collected from SPF chickens infected with highly pathogenic avian influenza virus A/turkey/Indiana/22-003707-003/2022 (H5N1). The results from our study showcased significant reductions in host rRNA levels for both depletion protocols, with TURBO DNase demonstrating superior performance. Regardless of the depletion assay used, both led to a reduction of not only host-specific but also bacterial reads, consequently enabling a notable increase in the number of sequenced viral reads. Specifically, in samples treated with TURBO DNase, viral reads constituted 7.7 and 3.1% of the total reads, whereas only 0.4 and 0.6% of viral reads were presented in untreated samples when sequenced on Nanopore and Illumina platforms, respectively. Moreover, a substantial 7-fold and 5-fold increase in the average number of viral reads per 100k sequences was observed in samples treated with TURBO DNase compared to those treated with DNase I on both platforms, correspondently. Our findings underscore the critical role of host and bacterial depletion for improved avian pathogen detection and advancing the field of avian disease research through NGS.