Recipient Organization
INSO BIOSCIENCES INC
237 TOWER RD RM 410
ITHACA,NY 14853
Performing Department
(N/A)
Non Technical Summary
Advancements in plant genomics have improved plant breeding efforts, but genomic sample preparation for the newest sequencing technologies (long-read sequencing)remains a key bottleneck. Preparation of plant genomes into high-molecular weight (HMW)DNA is more challenging than both mammalian sample preparation and short-read sample prep. Plant cells have cell walls that are not easily dissolved via chemical lysis, and plant matter often contains high levels of secondary metabolites, such as starchor polyphenols, which inhibit sequencing reactions and prevent isolation of larger DNA molecules. Long-read sequencing requires the extraction and purification of HMW DNA, a process still in its infancy relative to traditional genomic sample prep workflows, such as for standard shortread/Illumina sequencing. Thus, user frustration in sample prep remains a critical constraint to the growth of agrigenomics applications.Inso's technology will improve the efficiency of crop breeding efforts by U.S. agriscience. Food and agriculture account for nearly 20% of the U.S. economy,but America's role as a global leader in agriculture is threatened by a stagnation in public R&D expenditures. New agrigenomic tools could power crop improvements, but sample prep continues to create a bottleneck that prevents scalability. Our technology is poised to solve this by providing an automatable system for genomic analysis with the ability to handle high volumes of samples quickly while achieving reproducible, high-quality results. This will have wide-reaching impacts, improving U.S. food security and the health and welfare of the American public.
Animal Health Component
10%
Research Effort Categories
Basic
10%
Applied
10%
Developmental
80%
Goals / Objectives
The goal of thisproject is to address a key need in agricultural genomics (agrigenomics) for more efficient preparation of high-molecular-weight (HMW) plant DNA for long-read sequencing applications.Progressto utilize the benefits of long-read sequencing in agriculture applications have so far been constrained by the unique challenges posed by plant samples.Inso's microfluidicbased platform enables extraction and purification of high-molecular-weight (HMW) DNA for downstream genomic applications such as long-read sequencing. DNA sample prep is currently a critical bottleneck for plant genomic analyses for agricultural science R&D. By improving the quality, efficiency, speed, and cost-effectiveness of DNA sample preparation from plants for longread sequencing, Inso's technology will allow agriscience companies to develop new plant varieties that have higher crop yields, improved nutrients, and greater drought tolerance and disease and pest resistance. Inso's proposed system uses a novel microfluidic platform to extract and purify HMW DNA without the need for any specific surface chemistries. The system is scalable, automatable, and minimizes sample loss, hands-on time, and DNA damage, improving sequencing quality.To optimze the system for preparation of HMW DNA from plant cells for use in long-read sequencers, Inso's Phase I work involves the following objectives:1) Develop Protocol for Rapid Isolation of Nuclei from Plant Tissue in <30 Min, 2) Adapt DNA Tuning for Plant DNA Extractions to Yield >100kb Fragments, and 3) Validate DNA Prep Workflows by Achieving Sequencing N50 of 70kb+.This proof-of-concept project addresses a critical need in agrigenomics and, if successful, will support a Phase II project for further optimization of workflows and instrumentation development.
Project Methods
Objective 1: Develop Protocol for Rapid Isolation of Nuclei from Plant Tissue in <30 Min As plant cell walls are difficult to lyse chemically, we propose to include a pre-processing step into our plant workflows to extract the nuclei from the plant cells. The resulting suspension can then be loaded directly onto our chips. We will adapt a previous protocol 10 used to prepare plant nuclei for fluorescence-assisted cell sorting (FACS). The approach is straightforward and utilizes manual dissociation with a razor blade to release the nuclei from the plant tissue while maintaining nuclear integrity. However, this protocol requires optimization for our system due to differences in the desired output (and to ensure that the final process is user friendly for our customers). Dr. David Galbraith from the University of Arizona, who developed and published this method in Science, will act as a consultant on this project and provide input and advice for adapting this method for our system. We will start with the original buffer used in the published protocol since we have initial experience and success using this buffer.Optimization Experiments: We will grow young seedlings from at least three commercial crops that can be easily grown in lab such as maize, tomato, and pepper. Plant tissues will be harvested and used to test/optimize the nuclear extraction protocol. The parameters to be evaluated are shown in Table 1. Although several of these conditions likely interact with each other (for example, buffer cutting volume might influence cutting time), we will first test each parameter individually in isolation to get a better understanding of which factors appear to be the most important. Should further optimization be warranted, we will further study how multiple conditions may interact to influence the quality of the nuclear suspension to reach an optimal set of conditions.Evaluation: Suspensions will be prepared in triplicate and assessed by (1) confirming intact, mostly single nuclei suspensions by staining with an intercalating dye and then visualizing using fluorescent microscopy, (2) counting the stained nuclei using a hemocytometer, and (3) lysing a set volume of the suspension to measure DNA content using a fluorimeter.Success Metrics: Preparation of a plant nuclear suspension (must contain >1 μg DNA in <250 μL solution) in <30 min from ≤5 g plant tissue without specialized skill or equipment.buffer originally used with the published protocol, the following additional buffers can also be tested and parameters re-analyzed to identify a suitable protocol: MgSO4,LB01,Otto's buffer,Tris-MgCl2,and WPB.F.2 Objective 2: Adapt DNA Tuning for Plant DNA Extractions to Yield >100kb Fragments We have demonstrated the ability to selectively tune the desired sizes from anywhere between 10 kb to over 200 kb in length, demonstrated using human cell lines (see below). However, translation of protocols and the system's capabilities into processing plant material is non-trivial. Plant genomes pose a unique challenge in that there is great variability in genome size between species. For example, the smallest known plant genome is 63 Mb (Genlisea aurea) while the largest known plant genome is 148 Gb (Paris japonica), representing a 2350-fold range. Furthermore, chromosome sizes and ploidy numbers also vary across plant species, which could further influence the fragmentation process. Here, we will produce an optimized workflow for plant species. For evaluation, we will include plant species representing a range of genome sizes as well as those that are of significant agricultural importance, including: Paris japonica (148 Gb), maize (2.4 Gb), soybean (1.1 Gb), tomato (950 Mb), Arabidopsis (135 Mb), and Genlisea aurea (63 Mb). Optimization Experiments: We will grow seedlings and prepare nuclei suspensions for each of the above species according to our optimized protocol developed in Objective 1. We will then load the nuclear suspensions onto our chips and employ our established protocol for fragmenting human DNA as a starting point. Parameters will then be varied as outlined in Table 2. resolve DNA sizes up to 165 kb. For fragment sizes beyond 165 kb, we will use pulsed-field gel electrophoresis (PFGE), which can resolve fragments megabases in size.Success Metrics: Establish fragmentation protocols to enable consistent, size distributions with peaks at 20 kb for PacBio sequencing or >100 kb for Oxford Nanopore sequencing or BioNano optical mapping. Recover 500 ng to 1 μg of DNA per chip.Potential Pitfalls and Alternative Approaches: Should there be difficulties in reaching our success metrics for some or all of the plants, alternatives include testing different fragmentation enzymes (e.g., transposases, fragmentases, etc.) or making the microfluidic channels longer to accommodate and capture longer plant chromosomes.F.3 Objective 3: Validate DNA Prep Workflows by Achieving Sequencing N50 of 70kb+ We will prepare DNA using the optimized workflows developed in Objectives 1 and 2 and will analyze the resulting samples using Oxford Nanopore sequencing to validate the size and quality of the DNA. For our initial validation step, we will use maize, a crop of high interest to researchers, federal agencies, and agri-tech companies. A major hurdle in isolating HMW DNA from maize is removing the large amounts of starch from the DNA, which inhibits sequencing reactions and results in poor data quality. After this initial comparison, we will sequence other species tested in Objective 2 to determine how our system performs in removing different secondary metabolites and preparing DNA from different sized genomes.Evaluation: Plant DNA will be prepared using Inso's chip and our optimized workflows from Objectives 1 and 2. DNA prepared using standard kits (e.g., Circulomics NanoBind Plant Nuclei and NEB Monarch HMW DNA Extract) will be analyzed as controls, and all will be run in triplicate. We will perform sequencing with MinION and Flongle flow cells using both the rapid library prep kit and the ligation library prep kit. The following sequencing metrics will be evaluated for each sample: 1) read quality score, 2) N50 length, 3) mean read length, 4) output yield. Success Metrics: Inso's workflows produce superior results to controls, achieving at least a mean read quality >8 and an N50 length >70 kb for all plant species.Potential Pitfalls and Alternative Strategies: We have chosen to demonstrate proof of concept here using the Oxford Nanopore system, as this requires longer DNA fragments as input (>100 kb) compared to PacBio's sequencer (20 kb). Success will set a high bar and confirm our ability to extract and purify even the longest DNA fragments for analysis. However, an alternative is to use PacBio sequencing, which would be easier to achieve given the smaller fragment sizes needed and would still demonstrate proof of principle for the value of our technology for plant long-read sample prep. This has also been de-risked by our preliminary data showing that Inso's system results in better metrics following PacBio sequencing of human DNA.