The goal of this Phase I SBIR project is to demonstrate the feasibility of developing a new double-stranded RNA sequencing (dsRNAseq)-based virus screening platformthat will serve growers and nurseries of the U.S. horticulture industry. This goal will be supported by the following objectives:Objective 1:Determine virus detection limits of dsRNAseq;Compare the detection limit of RT-PCR and dsRNAseq for Little Cherry Virus 2 (LChV-2).To establish successful high-throughput sequencingassays, it will be important to determine the virus detection limits of dsRNAseq, and how these limits compare to those of standard, RT-PCR methods. We aim to give our clients the option to either run their samples as individual sequencing library preparations, or to run them as pools of multiple samples in a single library preparation. The latter is a desirable strategy for detecting the presence virus in a large orchard block or group of plants without having to screen each individual initially. Establishing detection limits will inform the maximum number of samples that can be pooled for screening at once. We aim to move toward screening large pools of samples whilst guaranteeing that a single infected sample in a pool of multiple samples could be detected. Detection limits of each diagnostic tool will be determined through comparing assays of pooled infected and healthy materials in a series of ratios (1:0, 1:10, 1:20, 1:50, and 0:1).Wewill also conduct comparative assays to determine what tissue types(e.g., leaf, bark, budwood, orin vitrotissue)are best suited for viral RNA extraction and detection.Objective 2:Optimize dsRNAseq workflow and data analysis; test infected and healthy materials from cherry, apple, and pear.Following establishment of detection limits, we will need to verify that that these limits are the same in multiple crops and determine optimal protocols to be used throughout the diagnostic pipeline, from sequencing to data analysis.We will optimize our workflow for dsRNAseq and downstream data analysis to establish a pipeline that can be rapidly utilized for analysis of different horticultural crops.Additionally, we will determine a downstream bioinformatics pipeline that will be used for sequence read processing, viral genome assembly, and taxonomic classification.Completion of Objective 2 will allow us to confidently sequence multiple barcoded samples containing dsRNA representing an array of known viruses in a single run and to successfully detect anyviruses present in all samples sequenced.