Project Number: 6040-32000-081-018-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Mar 1, 2022
End Date: Feb 28, 2025
Metagenomics Next Generation Sequencing (mNGS) targets to randomly sequence any nucleic acid in the sample. This allows mNGS to potentially identify all disease causing pathogens in a given sample. Oxford Nanopore Technology (ONT), a third generation sequencing platform provides extremely versatile and adaptable technology and presents a remarkable opportunity for mNGS approach to become an accessible diagnostic tool for veterinary diagnostics. The goal of this project is to utilize the broad spectrum of the metagenomics approach, but focus its powers towards poultry respiratory disease. We aim to increase mNGS sensitivity and depth specifically towards poultry respiratory pathogens. This can be achieved by combining the mNGS via ONT with the targeted enrichment of respiratory pathogens. We aim to establish an agnostic diagnostic approach that can used to test for all poultry respiratory pathogens at the same time. Validation of the generated results will be performed by comparing assay performance parameters to real-time PCR. Sequencing accuracy will be compared to Sanger or Illumina sequences. Outcomes of this project will be specific protocols, limits of detection and enrichment methods aiming to allow ONT to become a practical and useful poultry diagnostic tool. This project utilizes new technology in an applied approach to aid in maintaining healthy poultry and to preserve the nation’s food supply, which fits many of the program area priorities.
In testing clinical samples, one of the major impediments to higher sensitivity is that a high percentage of sequence reads are host sequences. Many commercial kits will reduce host and or bacterial rRNA sequences, and use a variety of approaches. Although effective, these approaches are expensive and are not targeted to poultry samples. We have recently demonstrated a more cost effective approach that allows selective targeting and depletion of host sequence which greatly increases the sensitivity to other pathogens. DNA probes, complementary to primarily to rRNA and select mitochondrial sequences host sequence, can be used to degrade host RNA using RNAse H treatment. The RNAse H treatment approach, which degrades the RNA in RNA/DNA hybrids is also much less expensive at about 10-12/sample as compared $35-50/sample for commercial kits. We intend to analyze each successive sequencing run to evaluate which host species are impacting sequencing results and an iterative approach to target additional sequences will be used. This approach will have diminishing returns because of the cost of adding additional sequences to the probe mix, but this approach will likely provide an increase in sensitivity for viral sequences. The same approach can be used to target bacterial rRNA sequences. This approach can also leave part of the 16s rRNA bacterial sequences untargeted to allow for their use of identification of important bacterial pathogens. Because bacterial rRNA sequences are more variable among the different phylum, additional iterative approach to deplete bacterial sequences will be used. In addition, trials using the non-selective amplification using Sequence-Independent Single-Primer Amplification (SISPA) approach will be performed and evaluated. These experiments will inform us about ways that can improve target read length, genome.