Progress 09/01/23 to 08/31/24
Outputs
Target Audience:The primary audience for our project includes farmers, nursery managers, and horticulture industry stakeholders who rely on accurate virus detection to maintain crop health and productivity. Viral pathogens pose significant risks to crop yields and quality, directly impacting economic stability in these sectors. Through this project, we aim to provide these groups with accessible, high-throughput diagnostics to manage virus threats proactively. Outreach to our target client base this year included participation in events such as the Washington State Horticulture Expo, the American Phytopathological Society annual meeting, and the Oregon State University Pre-Harvest Cherry Tour, where we presented and/or discussed the technology's benefits for virus detection. By addressing the needs of both large and small-scale growers, as well as academic and research communities, we are expanding awareness of advanced viral diagnostics methods to support agricultural sustainability while addressing an unmet market need. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training Activities In-House Training on ONT Sequencing:Team members received hands-on training on the Oxford Nanopore Technologies (ONT) GridION platform, which has allowed us to become proficient in library preparation, ONT sequencing, and data interpretation. As ONT technology and protocols continue to evolve, we receive ongoing support from ONT specialists, ensuring that our team remains current with the latest advancements and best practices. This ongoing training enhances team members' ability to manage high-throughput sequencing workflows independently, an essential skill as we transition exclusively to in-house ONT sequencing. Mentorship:Graduate students, junior researchers, and lab technicians on the project received mentorship from the PI, Co-PI, and Subaward PI, focusing on the technical aspects of sample preparation, RNA extraction, sequencing, and data analysis. Professional Development Activities Conference Participation:Attendance at the Washington State Horticulture Expo, APS annual meeting, and OSU Cherry Tour enabled team members to connect with industry professionals, share project insights, and gain knowledge on current plant health challenges. Workshops on Sequencing and Data Analysis:Staff participated in workshops on data analysis and sequencing methodologies, strengthening diagnostic accuracy skills. Texas A&M Collaboration:Students and interns gained hands-on experience in sequencing and pathogen diagnostics, advancing their knowledge in virus management. How have the results been disseminated to communities of interest?Conferences, Trade Shows, and Community Engagement To reach stakeholders and the public, we participated in conferences, trade shows, and local community events. These engagements allowed us to present our service as a whole, emphasizing its potential benefits and applications for various audiences. Partnerships Collaborations with industry and agricultural partners have allowed us to showcase the practical applications of our sequencing service in specific sectors. Online and Social Media Outreach We are continually updating our company website (www.qualterraag.com), as well as our social media pages on Facebook and LinkedIn, to share new developments, service offerings, and presentations related to our viral diagnostic platform. These online updates provide stakeholders and the public with timely information about project advancements, enhancing accessibility and engagement with our work. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will concentrate on further refining sequencing and barcoding protocols, supporting increased sample throughput, and strengthening quality control measures. While the primary R&D objectives and associated funding were largely fulfilled by August 31, 2024, remaining Technical and Business Assistance (TABA) funds necessitated a no-cost extension. This extension, with an adjusted end date of August 31, 2025, allows us to build upon our achievements, fine-tune processes, and ensure scalability as we transition toward broader deployment. Further Optimization of Barcoding Capacity and Sequencing Throughput? We will conduct additional testing with client samples to further define optimal barcoding configurations, balancing multiplexing with data accuracy to establish reliable capacity limits. We will also streamline sequencing and data workflows to support larger volumes, ensuring consistent data quality and operational efficiency. To support scaling, we will evaluate high-throughput processes to establish best practices for cost-effective, accurate sequencing at increased loads. Advanced Data Analysis for Sequencing Quality We will enhance data analysis tools and implement additional quality control measures to ensure data integrity, particularly when handling variable-quality RNA samples. These updates will strengthen our high-throughput diagnostic capacity while maintaining the accuracy essential for virus detection. One specific enhancement will be the development of a script to estimate relative viral titer. This tool will account for the amount of data generated in each sequencing run and the number of barcoded samples processed, allowing for reliable viral titer comparisons across different sequencing runs.Estimates of viral titer will be a valuable piece of information that we will provide to our clients. Expected Outcomes for Next Reporting Period By the end of this period, we expect to have further improved our barcoding and library preparation protocols, refined workflows to support high-throughput sequencing, and enhanced quality control measures. These developments will ensure our team is fully equipped to manage larger sequencing projects, providing reliable, scalable solutions as we expand our service offerings to meet growing client needs.
Impacts
What was accomplished under these goals?
Issue or Problem Addressed Crop viruses pose a significant threat to the horticulture industry, leading to economic losses. Current diagnostic methods can be costly and slow. This project aims to develop a fast, reliable, and cost-effective diagnostic platform for proactive sequencing-based virus management, reducing crop losses and contributing to the long-term sustainability of the horticulture industry. Intended Audience and Immediate Impact Primary beneficiaries include farmers and nurseries who depend on effective virus screening for crop health management. This diagnostic tool aids in early virus detection, helping to mitigate risks and enhance economic stability in the horticulture sector. The project's broader impact supports agricultural sustainability by providing essential tools to combat crop disease. Major Goals, Activities, Data Collected, and Results Objective 1: Optimize Protocols for Viral Detection To improve virus detection efficiency and accuracy, we focused on refining RNA extraction, mastering sequencing library preparation, and establishing sequencing protocols and best practices. In the early stages of the project, we explored automating RNA extraction using the Oktopure platform and identified several optimizations, including enhanced lid fittings to prevent sample contamination and the use of larger well plates to improve processing time. While these modifications significantly improve Oktopure's performance, manual extraction is currently providing the consistency needed (and meeting throughput requirements) to maintain high-quality results for sequencing.In lieu of increasing RNA extraction throughput, we focused on optimizing methods to extract high-quality RNA from the challenging samples we receive from growers. We refined both silica column-based and magnetic bead-based RNA extraction protocols to deliver clean RNA with reliable concentrations, ensuring consistent performance and rapid execution. To enhance efficiency during library construction, we introduced multichannel pipetting and strip tube processing, significantly reducing preparation time. Library construction now spans two days: RNA extraction, reverse transcription, and strand switching are completed on the first day to preserve quality, with samples stored at -20°C before final assembly. This approach is particularly effective with 8-12 samples, enabling high-quality libraries while maximizing multiplexing capacity. Using this method, one technician can prepare 50-60 libraries per week. Before sequencing, barcoded libraries are normalized and loaded in equimolar amounts, ensuring balanced inputs. This workflow provides sufficient time to evaluate and order libraries strategically, allowing low-yield cDNA libraries to be paired with higher-yield ones to optimize sequencing performance. Additionally, we conducted a comparative analysis of sequencing results using Illumina (both fully outsourced and in-house prep models) versus our in-house ONT GridION platform. This analysis demonstrated that ONT sequencing is more cost-effective and faster than the Illumina platform, without compromising detection accuracy.We demonstrated that in-houseONT GridION sequencing significantly reduces costs and turnaround times compared to Illumina platforms, addressing key challenges for our clients. Sequencing cores often required 5-6+ weeks to deliver results, which was inconvenient for growers making time-sensitive decisions. Moreover, data generated through in-house ONT sequencing proved more accurate, as comparative analyses revealed cross-contamination during automated library preparation at sequencing cores--an issue that, while acceptable for some applications, is problematic for virus detection. We have been able to mitigate this issue through carefully controlled in-house sample preparation, ensuring clean and reliable libraries. This shift ensures that we can consistently deliver faster, more reliable results tailored to the needs of our clients. In light of these findings, we have transitioned exclusively to in-house ONT GridION sequencing, allowing us to deliver faster and more affordable virus detection for growers, directly supporting their ability to manage virus threats proactively. Objective 2: Evaluate and Expand Multiplexing Capacity To support platform scalability, we evaluated the multiplexing capacity of the ONT GridION. While the rapid barcoding kit theoretically allows up to 96 samples per flow cell--equating to a maximum of 480 samples per sequencing run across the GridION's five flow cells--in practice, we have used the native barcoding kit, which supports 24 samples per flow cell. Testing sample sets of 1, 6, 12, 24, and 48 revealed that using 12 to 24 samples per flow cell achieves an optimal balance between throughput and virus detection sensitivity. Increased throughput is feasible; however, we have also found that the quality of starting RNA can significantly influence data quality from the sequencer. Lower quality RNA--which is commonly due to factors such as tissue type, season of sampling, and sample handling--requires generating more data per sample to maintain diagnostic accuracy. Our insights from multiple sequencing rounds and varying RNA quality have equipped us to make informed decisions on sample batching in real time, ensuring that each sequencing run maintains high standards of accuracy and efficiency. Outcomes and Impact of Accomplishments This project's accomplishments include creating a streamlined, scalable, and efficient virus detection platform that directly benefits the horticulture industry. By optimizing protocols and shifting to ONT sequencing, we have established that we can indeed reduce both costs and turnaround times, making broad-spectrum virus detection more accessible for growers and nurseries. This enhanced access to timely diagnostics enables them to identify and mitigate virus threats early, protecting crop health, reducing economic losses, and supporting sustainable agricultural practices.
Publications
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:The target audience includes members of the US horticulture industry. For Phase II, we are addressing the growers and nurseries of the fruit tree sector of theindustry, as well as hop and grape producers. In disseminating the progress and interimresults of our viral diagnostic platform development as well as future goals for this work at a regional, national, and international horticulture conferences, we have also garnered interest from producers of other crops. Changes/Problems:Delay in Equipment Acquisition/Training Due to unforeseen delays in purchasing our ONT sequencer and completing the Advanced User Training, it has taken several months beyond what we had originally projected to begin the ONT-related aspects of the work, including GridION library preparation and sequencing. We recently completed this training successfully and now anticipate no further delays in this aspect of the project. Troubleshooting Associated with Process Optimization As discussed in the accomplishments section, one of the areas in which we are optimizing our protocols for RNA extraction is process automation using our Oktopure robot. We are currently troubleshooting two issues with RNA extraction on the robot: 1.)the inability to quickly sample and freeze a 96-well block of samples and 2.) the inability to quickly grind, lyse, and mix the samples without risking cross contamination.The process of placing the ideal amount of freshly cut plant tissue into 96-well plate takes 5-7 hours; moreover, the 96-well plates and lids designed for the Oktopure currently do not fit tight enough to prevent cross contamination of samples.To address these issues, we have sourced 24 and 48 deep well plates with very tight and deep fitting silicone lids.This will give us the ability to process the sensitive initial steps faster as well as the option to use a chloroform step, if needed, to produce a higher quality initial extraction.Two to four of these plates can then be arrayed to a single 96-well plate to finish on the Oktopure robot.The sampling of tissue in 24-well plates will greatly improve this process; not only is it fewer samples at a time, but the wells are larger allowing tissue to be added to them much faster. It's important to note that the challenges with the Oktopure do not set us back to completion of the SBIR work (as we are experiencing no trouble with manual RNA extraction) incorporating automation into portions of our workflow will be important for enhancing efficiency in the future. What opportunities for training and professional development has the project provided?Thisproject has provided our team members with the chance to present the interim results and progress of this workat regional, national, and international conferences, enabling us to share findings and network with professionals in the field(both academic and ag-industry professionals). It has also allowed for the opportunity to gain trainingon the ONT GridION sequencing platform, equipping our team with the skills to operate this cutting-edge technology for crop diagnostics. Additionally, the project has facilitated training of graduate student internsin specialized areas such as plant molecular biology, next-generation sequencing technologies, and pathogen diagnostics in horticultural crops. Lastly, the project has facilitated a collaboration with Texas A&M University, a national leader in the realms of crop genetics and genomics research, thereby enhancing the breadth and depth of expertise available to the team. How have the results been disseminated to communities of interest?Interim results have been shared with the Clean Plant Center Northwest, horticulture industry members who have supported this work, and clients (who are primarily farmers and nurseries). Ongoing work and interim results were presented at the Northwest Hort Expo in Wenatchee, Washington;at the Plant and Animal Genome Conference in San Diego, California; and at the First International Symposium on Plant Propagation, Nursery Organization and Management for the Propagation of Certified Fruit Trees in Locorotondo, Italy. We are continually updating our company website (www.qualterraag.com), as well as social media pages (Facebook & LinkedIn), with new developments and news associated with our viral diagnostic platform and service offerings, as well as presentations related to our work. What do you plan to do during the next reporting period to accomplish the goals?As we have recently received our professional training by Oxford Nanopore Technologies on our new GridION sequencer, our efforts moving forward will focus on optimizing our protocols for in-house sequencing. Primarily, we will be conducting experiments to determine how many libraries we can prepare and individually barcode for sequencing together, as well as how many total barcoded samples we can run on a single flow cell (or across all 5 GridION flow cells). This information will allow us to answer several key questions including what the maximum throughput is for our platform, how much data is generated per sample, what theminimum cost per sample is when maximum throughput is reached.
Impacts
What was accomplished under these goals?
Objective 1: Protocol Optimization and Evaluation Many of the plant tissue samples that we have obtained for this SBIR work have been those we have directly collected, or received from, the Clean Plant Center Northwest. These have generally yielded high-quality RNA for sequencing, largely because of the strict protocols we adhere to in order to ensure the samples remain in good condition prior to entering our diagnostic pipeline. However, from a business standpoint, most of the samples we receive for diagnosis on our platform will be sent to us by our clients, most of whom are farmers. These samples may not be immediately refrigerated after the clients collect them, and they might not be shipped immediately after harvesting. Thus, a main area for protocol improvement has been optimizing nucleic acid extraction from samples that have not been maintained in optimal conditions before and during transportation.To address this, in the lab, we are experimenting with chemical preservatives, increasing our sampling capacity, adhering to a strict cold chain once samples reach our facility, and snap freezing samples immediately after sampling. Outside of the lab, we will provide clients with shipping kits with coolers, ice packs, and pre-paid express shipping labels. We will also work with our salesperson to inform our clients of best practices for scheduling incoming samples to have staff available to sample same day. We have also modified our RNA extraction procedures to increase RNA quality and yield, which has improved our ability to produce sequence quality RNA in low to medium throughput.We have also optimized the amount of starting tissue required for RNA extraction, adding additional chemicals to reduce the amount polyphenols and polysaccharides present as well as antifoaming agents to allow better homogenization of samples. For enhanced quality assessment of extracted RNA, we have begun utilizing the new RNA QC cartridge for our QiAxcel capillary gel electrophoresis system to check the RNA quality (RIN score), presence of contaminants and concentration of subunits. To facilitate in-house library prep, we have begun using the Zymo Research Zymo-Seq RiboFree Total RNA kit to construct libraries for outsourced illumine sequencing. In addition to the aforementioned protocol optimizations, a major update to the efficiency of our RNA extraction workflow has been the integration of our Oktopure automated nucleic acid extraction robot, which facilitates high-throughput RNA extraction. We are currently troubleshooting some aspects of automated extraction (see challenges section below); however, following optimization of this process, we expect to be able to prepare samples for sequencing at much higher throughput. Ongoing protocol optimization for RNA extraction, library prep, and ONT long-read sequencingis being supported in part by our subaward collaborators at TAMU. Illumina Library Preparation and Sequencing Ribosomal depletion is the current best method for limiting the amount of ribosomal RNA (rRNA) reads in RNA-seq data, thereby enriching the content of protein-coding nucleic acids, including viral materials.Most library kits have a ribosomal depletion option in addition to poly-A selection--we have conducted Illumina sequencing using both methods.We have had libraries constructed and sequenced at both WSU Sequencing Core and Texas A&M Sequencing Core (we have begun using the latter due to subaward PI Dhingra's affiliation--we are able to get better rates and turnaround time while still achieving high depth of sequencing). ONT Library Preparation and Sequencing We purchased the Oxford Nanopore GridION sequencer that will be important as we move forward with our work to improve our diagnostic efficiency, and we recently completed the advanced training process with a Nanopore sequencing specialist.We have successfully constructed several libraries; we are working to produce 6-8 barcode multiplexed libraries this month. Comparison of Results, Cost, and Turnaround Time of Different Platforms We have sequenced enough samples at WSU and TAMU, as well as in-house on our new GridION, to provide a preliminary cost, data generation, and turnaround time comparison (see below). The maximum number of samples we can multiplex will be established in our year 2 work. WSU Core Illumina HiSeq (Ribo-depleted) Cost per sample for ≥ 24 samples: $338.75 Reads per run: 380M Read Length: 2x100 bp Data per Run: 76 Gb Data per sample: ~3.17 Gb Turnaround Time: ≥ 6 weeks TAMU Core Illumina NovaSeq (Poly-A) Cost per sample for ≥ 24 samples: $229.74 Reads per run: 800M Read Length: 2x100 bp Data per Run: 160 Gb Data per sample: ~6.67 Gb Turnaround Time: ≥ 5 weeks GridION (Poly-A) Cost per sample: $198.95* Reads per run: 50M-150M Read Length: 500 bp - 20 kb (Variable) Data per Run: 250 Gb Data per sample: ~10.42 Gb Turnaround Time: Anticipate 2 weeks GridION (Ribo-depleted) Cost per sample: ~$200-300* Reads per run: 50M-150M Read Length: 500 bp - 20 kb (Variable) Data per Run: 250 Gb Data per sample: ~10.42 Gb Turnaround Time: Anticipate 2 weeks GridION (Direct RNA-seq) Cost per sample: ~$200-300* Reads per run: 50M-150M Read Length: 500 bp - 20 kb (Variable) Data per Run: 250 Gb Data per sample: ~10.42 Gb Turnaround Time: Anticipate 2 weeks *This does not account for the possibility of using recycled flow cells. Objective 2: Evaluation of MultiplexingCapacity We have successfully sequenced up to 40 samples on a single NovaSeq and Illumina HighSeq run with ample data to determine viral infection even down to several reads; this means that a minimum of 350 reads per sequencing run, respectively or about 9.5M reads per sample, has been sufficient to establish whether virus is present or absent in a given sample, and and allows us plan for future runs with multiplexed samples. In the future, it is of interest to multiplex additional samples, to establish at what point we should limit the number we include in a single sequencing run. As we only recently received our training on the ONT GridION sequencer, we have not yet barcoded multiplexed samples in-house; however, this is work we are planning to begin early in Year 2, with the next step being to multiplex 8 to 24 samples on a single GridION flow cell and determine the optimal number of samples that we can sequence on a flow cell while still generating enough data for each.We will run both poly-a and ribo-depleated ONT libraries to assess the number of usable reads generated by each method.
Publications
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