Next-Generation Sequencing for the Detection of Microbial Agents in Avian Clinical Samples

Abstract

Direct-targeted next-generation sequencing (tNGS), with its undoubtedly superior diagnostic capacity over real-time PCR (RT-PCR), and direct-non-targeted NGS (ntNGS), with its higher capacity to identify and characterize multiple agents, are both likely to become diagnostic methods of choice in the future. tNGS is a rapid and sensitive method for precise characterization of suspected agents. ntNGS, also known as agnostic diagnosis, does not require a hypothesis and has been used to identify unsuspected infections in clinical samples. Implemented in the form of multiplexed total DNA metagenomics or as total RNA sequencing, the approach produces comprehensive and actionable reports that allow semi-quantitative identification of most of the agents present in respiratory, cloacal, and tissue samples. The diagnostic benefits of the use of direct tNGS and ntNGS are high specificity, compatibility with different types of clinical samples (fresh, frozen, FTA cards, and paraffin-embedded), production of nearly complete infection profiles (viruses, bacteria, fungus, and parasites), production of “semi-quantitative” information, direct agent genotyping, and infectious agent mutational information. The achievements of NGS in terms of diagnosing poultry problems are described here, along with future applications. Multiplexing, development of standard operating procedures, robotics, sequencing kits, automated bioinformatics, cloud computing, and artificial intelligence (AI) are disciplines converging toward the use of this technology for active surveillance in poultry farms. Other advances in human and veterinary NGS sequencing are likely to be adaptable to avian species in the future.

Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice

“…49 Assays can be multiplexed to screen for many infectious agents in one run. 1,23 In addition to its value for detection and identification of pathogenic organisms, NGS can be used to examine genes for antibiotic resistance and to type organisms for understanding transmission and epidemiology of an outbreak. 8,20 Here we employed targeted NGS to speciate the member of ECC we initially isolated by culture.…”

Non-Targeted RNA Sequencing: Towards the Development of Universal Clinical Diagnosis Methods for Human and Veterinary Infectious Diseases

“…Sample preservation and storage, shipping, and extraction are likely to significantly influence results. Afonso's 2023 review also provides a valuable resource outlining critical aspects of sample collection (Table 1) [43]. during sample processing.…”