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Bacterial Amplicon Subtyping


surveillance. For example, PulseNet, a national subtyping network of over 80 labs nationally and over 80 international laboratories, tracks foodborne outbreaks and currently relies on isolates for generating molecular fingerprints for foodborne bacterial surveillance for over 90,000 isolates.
In response to these new challenges, this proposal aims to develop new molecular fingerprinting techniques that can be used with specimens such as stool. The assay approach will rely on PCR amplification of informative regions and sequencing of amplicons using short read technology such as the Illumina platforms. An algorithm for the identification of heterogeneous regions for isolate subtyping and design of conserved flanking primers is necessary for the development of these assays, but are currently not available commercially or in the open source community. Providing an algorithm for development of these primers, or the primers generated from the algorithm, would be of great use to the public health community and could be used in both public health laboratories nationally and internationally as well as infection control groups in a health care setting.
Project Goals
The offeror will provide an innovative bioinformatics algorithm in their software platform that allows the user to design amplicons that can be sequenced to determine the subtype of a pathogen. Specifically, the software must identify

heterogeneous regions which are useful for strain typing and also flanked by conserved sites suitable for primers. The resolution of strain subtyping must be equivalent to current WGS-based subtyping techniques for isolates and can distinguish isolates associated with an outbreak from background cases. The algorithm must include an option to measure and adjust the amount of heterogeneity so that the user may decide how much is needed for subtyping. The software must identify these regions either automatically (defining subgroups naturally) or allow the user to pre-define the groups or subtypes for which the primers will be designed and include the ability to consider Illumina or other sequencing adapters and multiplex barcodes when testing for primer-to-primer interactions. Ideally, the software must be packaged so that it could work directly with existing infrastructure such as high-performance computing (HPC) resources.
Phase I Activities and Expected Deliverables
The contractor will design the algorithm to meet the above specifications and perform in silico validation of amplicons. The contractor will use epidemiologically relevant sequence data from the relevant pathogen groups for testing their algorithm and will be expected to provide preliminary results upon completion of phase I. The results must include primers, fasta files for amplicons, in silico PCR results, annotated bedgraphs and coverage histograms.
For Successful Phase I Awardees ONLY (Expected Phase II Deliverables)
In Phase II, the contractor will further refine the algorithm and primers based on laboratory results. The contractor will also evaluate the usefulness of the software for developing assays for generating molecular fingerprints of pathogens. At the completion of Phase II, the contractor will provide a stand-alone license for a single copy of the software which is a compiled, command-line binary that runs on the high performance computing clusters and standalone Linux workstations installed with Ubuntu; sample configuration files and documentation must be included in the binary install. With this software, the developers must create training documentation that instructs the user in how to operate the software to generate primers in conserved regions that flank heterogeneous regions and generate the output outlined in the Activities and Expected Deliverables section above.
This novel bioinformatics approach would enable public health professionals to identify phylogenetically informative regions and design rapid PCR subtyping approaches that detect and subtype pathogens directly from disease state stool and other specimen types. In addition, this approach will significantly improve assay development strategies by incorporating multiple steps to assay design into a single, streamlined platform. Furthermore, the assays that are developed will be deployed in public health labs worldwide, allowing for continued surveillance of these pathogens and the identification of outbreaks in the absence of cultures.
Commercialization Potential
Currently there are no software programs on the market that design primers around heterogeneous regions in bacterial genomes for a sequencing-based subtyping workflow. The addition of this product to the subtyping market is necessary now that CIDT tests are being more widely used not just for identifying pathogens associated with foodborne infections but also febrile illnesses, respiratory illnesses, and other disease types. Due to the rapid adoption of CIDTs in the health sector, the design of novel and innovative approaches for subtyping directly from specimens is needed within the public health surveillance community. With the product the awardee designs, subtyping can begin with the same specimen on which a CIDT test is performed, rather than the isolate which can take several days to weeks to culture. By being able to more rapidly subtype pathogens, outbreaks are detected sooner which means the public can be alerted to health threats sooner and more lives saved. This software product or the primers designed using this product would be of interest to public health professionals and those in the health care sector that need to identify related illnesses through subtyping to detect and stop outbreaks.

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