Progress 07/01/21 to 02/28/22

Outputs
Target Audience:Target Audience: The target audience includes members ofthe US horticulture industry. For Phase I, we are addressing the growers and nurseries of thefruit tree sector of theindustry. Indisseminating preliminary results of our viral diagnostic platform development as well asfuture goals for this work at a regional horticulture conference, we have also garnered interest from other sectors, including the hops, grapevine, and hazelnut industry, to name a few. Efforts: Thus far, efforts have included developing andassessing feasibility of a viral enrichment-based sequencing platform for pathogen diagnostics; documenting the resulting outcomes and next steps; coordinating with the subaward institution regarding sequencing data analysis;training a Graduate Research Intern; and extension and out reach at an annual horticulture meeting and via interviews with Washington State magazine and a university newspaper. Changes/Problems:While we hypothesizedthat dsRNA enrichment would be the best method for optimizing sequencing-based virus detection, we have established that our back-up strategy of using ribosomal depletion to enrich for viral material is actually more feasible and efficient. Thus, we needed to modify our strategy for viral enrichment while retaining the essence of our objectives.We intend to include a detailed summary of our efforts using the dsRNA method, as well as the reasoning behind why ribosomal depletion-based enrichment will be the best pre-sequencing method moving forward,in our Phase II proposal. Ultimately, the outcome of successful viral enrichment (regardless of the strategy used to acheive it) is expected to facilitate more efficient and early detection of viral infections in crops. Another challenge we have encountered is that, due to the pandemic, turnaround time for RNA sequencingat theWSU Genomics Corehas been longer than expected. This has meant that most of our sequencing work is being completed towards the end of the project. For the future, we are assessing other alternatives, including purchasing our own sequencing unit or using another sequencing center,to be able to acheve the goal of providing timely horticultural crop diagnostics at a competitive price. Ultimately, we would like to do all of our library preparation and sequencing in-house. What opportunities for training and professional development has the project provided?The PI and Co-PI have had the opportunity to advance their expetise insequencing and PCR-detection levels of plant pathogens, specifically for horticultural crops.The knowledge they have gained, and the applications of this knowledge, set them upto advance the technology further and to be competitive as molecular biologists and genomic data analysts in the horticulture industry. The PI has also honed her skillset in genomic data analysis; through collaboration with the subaward institution, she has advanced her knowledge of how to utilize genomics software programs like CLC genomics workbench, which will be critical for the future of this project. In addition to the training and professional development opportunities this project has afforded the project leads, the Co-PI had the opportunity to train a Graduate Research Intern in the processes of plant RNA isolation, PCR-based diagnostics, communication with consulting groups (like the genomics core). The Co-PI also had the opportunity to network with industry members and share preliminary results of our sequencing-based diagnostic platform at the Washington State Tree Fruit Association's annual meeting.? How have the results been disseminated to communities of interest?The co-PI attended the Washington State Tree Fruit Association's annualmeeting where she networked with industry members and shared the status of the RNAseq-based viral detection methods that are in NuPhY's pipeline. The present work has been well received and has generated a great deal of interest from the industry, which has led to follow-up conversations and project planning. Additionally, the PI and NuPhY's CSO engaged in two interviews, one with the Washington State Magazine and the other with the Daily Evergreen (WSU Newspaper) to share the goals for the high-throughput sequencing-based pathogen diagnostics in the horticulture industry and to disseminate results of the work thus far. The Washington State Magazine interview was published in the Winter 2021 issue, and the Daily Evergreen interview is currently in press. What do you plan to do during the next reporting period to accomplish the goals?We will complete data analysis and results summary from the initial round of sequencing. In the coming weeks, we have some additional samples to send to sequencingwhich will facilitate more precise determination ofthe point at which RT-qPCR and RNAseq (ribosomal depleted and standard) differ in detection limits. We are in the process of isolating RNA for this last round of sequencing and expect to have the data ready to analyze by the third week of February. By the time of the next report,will have completed a comprehensive summary of the outcomes and next steps for our Phase II application.

Impacts
What was accomplished under these goals? Objective 1: We found that our back-up strategy ofribosomal depletion-based enrichment (rather than dsRNA enrichment)is the best strategy for viral enrichment for sequencing moving forward. We are currently in the process of establishing detection limits of ribosomal RNA depletion-based sequencing compared to standard RNA sequencing and PCR in different tissue types (leaf, bark, midrib), using Little Cherry Virus 2 as a case study. To do this, we produced dilution series with the highest dilution factor being 1:500 (infected-clean), based on the limits we established for PCR-based detection. One half of the diluted material was subjected to ribosomal depletion prior to sequencing and the other half was not. We have just received the sequencing results back from the WSU genomics core and are now in the process of analyzing them in conjunction with the subaward institution. Objective 2: As planned, we have sequenced infected materials (containing multiple, known viruses) from cherry, apple, and pear. We have just received the sequencing results back from the WSU genomics core and are now in the process of analyzing them in conjunction with the subaward institution. We will have results to report on this objectiveby the end of the month.

Publications

Progress 07/01/21 to 02/28/22

Outputs
Target Audience:Target Audience: The current target audience includes members of the US horticulture industry, although ultimately we can expand our technology to serve any crop production industry. For Phase I, we addressed the growers and nurseries of the fruit tree sector of the horticulture industry. In disseminating preliminary results of our viral diagnostic platform development as well as future goals for this work at a regional horticulture conference, we have also garnered interest from other sectors, including hops, hemp, grapevine, and hazelnut industries, to name a few. Efforts: We developed, and established feasibility of, a viral enrichment-based sequencing platform for pathogen diagnostics; documentedthe resulting outcomes and next steps; coordinatedwith the subaward institution regarding sequencing data analysis; traineda Graduate Research Intern; and particpated in extension and outreach at an annual horticulture meeting, as well as via interviews with Washington State magazine and a university newspaper. We summarized the Phase I efforts in detail in our Phase II application. Changes/Problems:As indicated by the title of our Phase I proposal and in the original Phase I objectives, our Plan A was to conduct the pre-sequencing viral enrichment step by selectively increasing the concentration of double-stranded RNA (dsRNA) in the samples--the logic being that dsRNA is the form that actively replicating viral genomic material takes. We found that the dsRNA extraction kit that we had proposed to use was no longer in production. Moreover, an alternative kit was available, which we tested. However, we found that this kit was sub-optimal for several reasons. First, it required a PCR amplification step post dsRNA extraction, which further extended the time and labor to process the samples. Second, and most importantly, it yielded extremely low quantities of genetic material that were not conducive to sequencing library preparation; while we were able to sequence dsRNA libraries, achieving library preparations of sufficient quality to sequence was a major challenge. Because of these limitations, we concluded that dsRNA enrichment was not the ideal strategy, and we proceeded with our alternative. Our Plan B strategy (which we had also specified in the Phase I proposal) of viral enrichment via depletion of plant ribosomal RNA prior to HTS proved to facilitate far greater sensitivity of detection than dsRNA enrichment, and thus, we adjusted our methodology accordingly for the remainder of our Phase I work (also see section on goal accomplishments). What opportunities for training and professional development has the project provided?The PI and Co-PI have had the opportunity to advance their expertise in sequencing and PCR-detection levels of plant pathogens, specifically for horticultural crops. The knowledge they have gained, and the applications of this knowledge, set them up to advance the technology further and to be competitive as molecular biologists and genomic data analysts in the horticulture industry. The PI has also honed her skillset in genomic data analysis; through collaboration with the subaward institution, she has advanced her knowledge of how to utilize genomics software programs like CLC genomics workbench, which will be critical for the future of this project. In addition to the training and professional development opportunities this project has afforded the project leads, the Co-PI had the opportunity to train a Graduate Research Intern in the processes of plant RNA isolation, PCR-based diagnostics, communication with consulting groups (like the genomics core). The Co-PI also had the opportunity to network with industry members and share preliminary results of our sequencing-based diagnostic platform at the Washington State Tree Fruit Association's annual meeting. Another major opportunity for training and professional development that this project provided was the TABA services provided by the Larta Institute. We worked with our principal advisor to develop a compelling commercialization plan, and are in ongoing communication with a Marketing and Branding Specialist and a Customer Discovery Framework Specialist. These services have been instrumental in our understanding of how best to advance our service platform beyond the R&D stage. How have the results been disseminated to communities of interest?The co-PI attended the Washington State Tree Fruit Association's annual meeting where she networked with industry members and shared the status of the RNAseq-based viral detection methods that are in NuPhY's pipeline. The present work has been well received and has generated a great deal of interest from the industry, which has led to follow-up conversations and project planning. Additionally, the PI and NuPhY's CSO engaged in two interviews, one with the Washington State Magazine and the other with the Daily Evergreen (WSU Newspaper) to share the goals for the high-throughput sequencing-based pathogen diagnostics in the horticulture industry and to disseminate results of the work thus far. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Results from Technical Objective 1: 1.) Rather than dsRNAseq based enrichment, ribosomal depletion-based enrichment proved a more effective strategyin the context of our platform. Prior to beginning our Phase I R&D, we expected that viral double-stranded (ds)RNA enrichment would be the best strategy for maximizing viral genetic material present in RNA samples prior to sequencing, as it would entail removal of the most non-viral genomic material prior to sequencing library preparation. This was not the case, with our dsRNA enrichment methods proving suboptimal (see challenges section). However,our secondary, ribosomal depletion strategy was successful. Ribosomal RNA represents a majority of plant genetic material in the samples; by removing this material, we effectively reduced the amount of sequenced material by approximately 90%, thereby facilitating a larger data allocation to any viral materials present. Unlike the dsRNA approach, this strategy can be done in conjunction with the library preparation step and, thus, it is more efficient with regards to time, labor, and cost. 2.) Using Little Cherry Virus 2 (LChV-2) as a case study, we identified the viral detection limits of ribosomal depletion-aided RNAseq versus RT-qPCR, determining that our viral enrichment and sequencing-based strategy is extremely sensitive. We also compared the detection limits of the dsRNAseq, as well as to those of standard RNAseq (without the precursor ribosomal depletion step).To establish detection limits, we generated a dilution series to identify the point at which each method was no longer able to detect a small amount of viral genetic material in the midst of a large amount of uninfected plant RNA. In order to reduce detection capacity enough to compare each screening method, we found that an extremely high dilution factor was required (up to 1,000 million x). While RT-qPCR detected virus in infected samples diluted up to 1 million times the starting concentration with clean RNA, RNAseq with the preceding ribosomal depletion step facilitated detection in infected samples diluted up to 100 million times with clean RNA. At a dilution of 1000x, both standard RNAseq (no viral enrichment step prior) and dsRNAseq were no longer able to detect the viral infection. Our alternate approach improved detection limit by two orders of magnitude over that of RT-qPCR. Overall, these results show that the capacity to detect viral material, even in highly diluted samples, is far superior in the samples subjected to viral enrichment via ribosomal RNA depletion prior to RNAseq. With this information, we can now confidently provide our clients with 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. 3.) In assessing viral detection capacity and the robustness of our platform for LChV-2 in different tissues, we determined that leaf tissue is the optimal tissue type for diagnostic analysis, but infections can be successfully detected in other tissue types. Consistent with our results above, the viral infection was easily detected via our enrichment aided HTS platform in all tissues; however, the leaf sample displayed the highest number of mapped sequencing reads, followed by midrib, and then by bark. Even though the leaf proved to be the best tissue in which to sensitively detect virus, we now know that we can still detect viruses at low titer in other structures at times of the year when leaves are not present; we will just need to be cognizant of the fact that detection limits are reached sooner in bark, and reducing the number of samples sequenced together in a run can facilitate greater data distribution to this low-titer viral genomic material. Results from Phase I Technical Objective 2: 1.) In sequencing infected materials containing multiple, known viruses from cherry, apple, and pear tissues, we successfully established that the detection capacity for RNAseq identified in Technical Objective 1 are comparable across different crops infected with a variety of different viruses.To establish that our screening platform is crop agnostic, infected cherry, apple, and pear leaf tissues were obtained from Dr. Scott Harper at the Clean Plant Center Northwest. The collected samples were previously confirmed by CPCNW to be infected with severalviruses. During the collection of these infected samples, Dr. Harper indicated that additional viruses/viroids were thought to be present beyond those that had been confirmed; thus, we expected sequencing to reveal these additional pathogens.For each crop, sequencing successfully detected all viruses that the CPCNW had documented to be present in the infected samples, as well as several previously unknown viruses. Many of the viruses detected by sequencing that were not known to be present prior to our high-throughput screening are not viral pathogens that are commonly associated with apple, cherry, or pear (e.g., hop and citrus viruses/viroids were detected). There are a couple of important takeaways from this finding. The first is that this outcome is a testament to the ease by which viruses spread--when samples were collected from CPCNW, many infected plants from multiple crop species in addition to apple and pear were quarantined together in the same room, providing ample opportunity for viral transmission between plants, (via insect vectors, human vectors, air circulation, or direct plant-to-plant contact). The second takeaway is that if standard methods for detection are used, and only viruses of primary interest targeted, there is the potential to misdiagnose a virus infected plant as clean, simply because the viruses of interest were not detected by a targeted RT-qPCR assay. Thus, these findings are an important demonstration of why comprehensive sequencing-based detection is necessary to provide an appropriate diagnosis of horticultural crop infection status. 2.) As part of Technical Objective 2, our collaborators at WSU aided the establishment of a downstream bioinformatics pipeline that we will use moving forward for sequence read processing and analysis in our viral diagnostic work.In assessing a number of software programs for detection of viral sequences as well as for general sequencing data analysis, both open-source and subscription-based, they determined that the Qiagen CLC Genomics Workbench suite provided excellent tools for sequence read mapping and analysis that are both user-friendly and widely applicable to many applications beyond just virus detection. Thus, this will be a critical tool for viral diagnostics and any other sequencing-based work moving forward, including whole genome-assembly based work that may be necessary if any novel viruses are discovered in the process of diagnosing infections in our clients' crops.

Publications