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Development of a Benchtop Laboratory Platform for Amplicon Deep Sequencing

Description:

Fast-Track proposals will not be accepted.
Number of anticipated awards: 1
Budget (total costs):
Phase I: up to $150,000 for up to 6 months
PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED.
Background
Molecular identification of infectious agents most commonly requires nucleic acid extraction, PCR amplification, and sequencing. Despite the availability of automated platforms, most standard laboratory protocols rely on manual procedures. Importantly, exposure to infectious materials during manual processing is a major safety concern, particularly when processing highly infectious agents. Moreover, manual processing affects reproducibility of testing, reduces throughput and increases sample processing time; as a consequence, time to accurate identification is delayed. Manual processing also introduces an increased risk of contamination which may confound results by the amplification of contaminating nucleic acids many fold during PCR processing. Thus, the implementation of a fully automated, closed platform is highly desirable for the efficient molecular identification of infectious agents, including hepatotropic viruses and other enteric, airborne and blood-borne pathogens. The CDC Division of Viral Hepatitis has designed and built an automated platform for the construction of DNA libraries from clinical samples in a compound fashion suitable for amplicon deep sequencing using the popular MiSeq instrument. Whereas capable of high throughput sample processing, this workstation is expensive and has a relatively large footprint. These characteristics impose limitations for the commercialization of a workstation suitable for smaller laboratories and clinical facilities. Thus, there is an important, and increasingly growing need for significantly smaller, ideally benchtop, comprehensive workstations capable of performing the same compound processes at a considerably reduced cost, yet exhibiting comparable biosafety standards and quality results. Whereas a number of units are commercially available for each of the individual steps of the compound process, there are no existing platforms capable of performing the entire process from A to Z in an automated, compound manner.
Project Goals
The aim of this proposal is to develop and evaluate a new, small footprint, benchtop automated nucleic acid extraction, amplification and sequencing system that fundamentally improves laboratory safety and quality control (QC). This platform consolidates all manual laboratory operations related to next-Generation Sequencing into a single compound process performed automatically by a single workstation in a small footprint, ideally at a reduced cost. The workstation should be

capable of being used in support of clinical testing, surveillance programs and outbreak investigations conducted by clinical and public health laboratories using NGS, which recently became a mainstay technology for the detection of pathogen drug resistance and transmission networks enabling public health interventions and management of patients in clinical settings. The workstation should be capable of performing RNA/DNA extraction from clinical samples, RT-PCR, nested PCR/tagging, amplicon clean up, quantification and pooling, resulting in DNA libraries ready for amplicon deep sequencing using the MiSeq platform. Whereas assembling workstations by integrating existing liquid handling robotic stations with stand-alone thermal cyclers, spectrophotometers and cappers/decappers (for specimen aliquoting) is possible, the resulting process is prone to all of the negative characteristics of manual processing outlined above. The goal of this project is to devise a new, affordable instrument that can perform all necessary functions starting from receipt of biological samples (such as whole blood, plasma, serum, stool, sputum and other) to construction of DNA libraries. This novel platform would be expected to significantly reduce constraints for established complex molecular next-generation sequencing-based methods. It should also contribute to strengthening quality control and biosafety in clinical and public health laboratories. This workstation will be capable of preparing DNA libraries for different pathogens and will require no manual steps except for the initial setup of the instrument for loading reagents/kits and clinical specimens. The unit is expected to be sufficiently flexible to accommodate different laboratory protocols. It should be readily adaptable to: (1) generate DNA libraries for the MiSeq illumina; (2) handle biological specimens such as whole blood, plasma and serum; (3) handle variable numbers of specimens from low to medium throughput; (4) use specific/customized reagents and kits for different pathogens. Availability of various preloaded programs for specific processing of specimens from different pathogens is also desirable. In such cases, user input should allow the incorporation of specific conditions (pathogen, number of samples, etc.) into the workstation controller to allow specific conditions from run to run. Substantial modifications and new programs must be registered in the workstation for quality control management in clinical and public health laboratories. Handling of laboratory protocols by the workstation should be highly reproducible and accurate to comply with clinical test requirements and automatic reporting on conducted tests should be outputted and available to managers and accounts with elevated privileges.
Phase I Activities and Expected Deliverables
During Phase I, the unit design and industrial diagrams with the final layout will be generated. Computer modeling of the final design is required for testing virtual laboratory protocols and fine tuning of individual processes and steps. The workstation is expected to perform the entire laboratory protocol starting from clinical samples to the NGS library within a few hours (between 8-12 hours) without any user intervention. It will be easily programmable, accommodate different specimen types (whole blood, plasma, and serum), variable numbers of specimens and sample volumes. It will be controlled by computer programs, which may be initiated manually or automatically using barcoded reagent kits. For enhanced QC, workstation data will be used to automatically detect instrument errors and control instrument maintenance.
For Successful Phase I Awardees ONLY (Expected Phase II deliverables)
For Phase II, full development and assembly of the pilot workstation is expected. It is important that the company awarded the contract demonstrate feasibility of performing all of the required steps to convert the prototype into a full-fledged platform. As aforementioned, the workstation is expected to be suitable for processing clinical samples for identification of pathogens. The platform will be extensively tested for its performance using serum panels specifically developed from specimens collected from individuals infected with hepatitis C viruses. Once developed, the workstation is expected to be thoroughly evaluated for use with other pathogens as well. Robustness of the automated workstation will be evaluated in comparison with currently established laboratory gold standards. Each step of the process will be assessed for reproducibility, accuracy, sensitivity, specificity, potential for cross-contamination, yield, time-to-run, safety, and throughput. Another criterion is the long-term stability of performance during continuous use. A full biosafety evaluation of the unit will have to be performed. Assessment of aerosols and user exposure will be conducted. Safety guidelines and recommendations will be put forward. This unit is expected to have a large market for state health laboratories and clinical laboratories, as well as reference laboratories worldwide.
Impact
Implementation of the workstation allows for continuous monitoring and evaluation of data, minimizing human errors and significantly improving accuracy of clinical testing and surveillance. It will significantly reduce hands-on time and exposure to infectious materials, thus fundamentally improving safety of laboratory work. The instrument is expected to have a broad use in public health and clinical laboratories. It is expected that the workstation will significantly improve quality of testing and accelerate sample processing for the identification of infectious agents and for outbreak investigations and molecular surveillance of different infectious agents. Both clinical and public health laboratories are expected to benefit from a commercially available fully automated, inexpensive, benchtop workstation for performing NGS. It will significantly reduce

complexity of the laboratory process for technical personnel in clinical and public health laboratories, while cardinally improving throughput and reproducibility of testing, reducing contamination, and keeping the cost per tested specimen low. Availability of the workstation makes massive and complex genetic testing as defined by Advanced Molecular Detection affordable and attainable for many laboratories in the United States and worldwide, fundamentally improving outbreak investigations, public health surveillance, and the identification and treatment of infectious diseases.
Commercialization Potential
The laboratory workstation is expected to have a very large market owing to a significant and rapidly growing need for complex genetic identification and testing of infectious agents for patient management in clinical settings (drug and antibiotic resistance) and for devising public health interventions to control viral and bacterial diseases. Each clinical and public health laboratory that is expected to conduct genetic testing and identification but lacks equipment and personnel capable of handling the demands of NGS is a potential customer. Integration of liquid handling platforms with many instruments is currently used to manage such tasks. However, a prohibitive cost, large footprint and complexity of the integrated assemblies reduce their application. A single, affordable, standalone instrument with a small footprint, specifically designed for performing all laboratory procedures required for NGS of different pathogens in a completely automated, closed mode will be useful to many laboratories in the United States and worldwide.

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