HR001118S0025.pdf

Broad Agency Announcement Friend or Foe

BIOLOGICAL TECHNOLOGIES OFFICE

HR001118S0025

February 16, 2018

HR001118S0025, Friend or Foe

TABLE OF CONTENTS

PART I: OVERVIEW INFORMATION

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

1.1. Program Overview

1.2. Technical Approach and Schedule

1.3. Program Milestones, Metrics, and Deliverables

1.4. General Requirements

2. Award Information

2.1. General Award Information

2.2. Fundamental Research

3. Eligibility Information

3.1. Eligible Applicants

3.2. Organizational Conflicts of Interest

3.3. Cost Sharing/Matching

4. Application and Submission Information

4.1. Address to Request Application Package

4.2. Content and Form of Application Submission

4.3. Funding Restrictions

4.4. Other Submission Requirements

5. Application Review Information

5.1. Evaluation Criteria

5.2. Review of proposals

6. Award Administration Information

6.1. Selection Notices

6.2. Administrative and National Policy Requirements

6.3. Reporting

6.4. Electronic Systems

7. Agency Contacts

8. Other Information

9. APPENDIX 1 – Volume II checklist

PART I: OVERVIEW INFORMATION

Federal Agency Name – Defense Advanced Research Projects Agency (DARPA), Biological Technologies Office

Funding Opportunity Title – Friend or Foe Announcement Type – Initial Announcement Funding Opportunity Number – HR001118S0025 Catalog of Federal Domestic Assistance Numbers (CFDA) – 12.910 Research and Technology Development Dates o Posting Date – February 16, 2018 o Proposal Abstract Due Date and Time – March 14, 2018 4:00 PM ET o Proposal Due Date and Time – April 25, 2018 4:00 PM ET o BAA Closing Date – April 25, 2018 o Proposers Day – February 28, 2018 https://www.fbo.gov/spg/ODA/DARPA/CMO/DARPA-SN-18-28/listing.html

Concise description of the funding opportunity - Friend or Foe will develop a high-throughput screening platform that improves our readiness against new microbial threats through isolation and phenotypic characterization of bacterial pathogenicity.

Anticipated individual awards - Multiple awards are anticipated.

Types of instruments that may be awarded - Cooperative agreement, procurement contract and other transaction.

Agency contact o Points of Contact The BAA Coordinator for this effort may be reached at:

FriendorFoe@darpa.mil

DARPA/BTO

ATTN: HR001118S0025

675 North Randolph Street Arlington, VA 22203-2114 https://www.fbo.gov/spg/ODA/DARPA/CMO/DARPA-SN-18-28/listing.html mailto:FriendorFoe@darpa.mil

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

This publication constitutes a Broad Agency Announcement (BAA) as contemplated in Federal Acquisition Regulation (FAR) 6.102(d)(2) and 35.016 and 2 CFR § 200.203. Any resultant award negotiations will follow all pertinent law and regulation, and any negotiations and/or awards for procurement contracts will use procedures under FAR 15.4, Contract Pricing, as specified in the BAA.

The Defense Advanced Research Projects Agency (DARPA) is soliciting innovative proposals to enable the development of technology for rapid phenotype-based identification of bacterial pathogens in complex environments. Proposed research should represent revolutionary advances in the science, devices, and systems needed for the isolation and characterization of potentially unknown and unculturable bacteria. Specifically excluded is research that primarily results in incremental improvements to the existing state of practice.

1.1. Program Overview

The Friend or Foe program will develop the capability to rapidly identify potential bacterial pathogens in complex environments through analysis of their behavior (i.e., their phenotype).

New pathogens, both naturally occurring and adversary-engineered, are increasingly likely to emerge due to changes in the environment, rising global population, and the wide availability of genetic engineering tools to both state and non-state actors. These factors, coupled with faster potential dispersal due to increasing global travel and population density, have significantly increased the danger posed by bacterial pathogens. Current detection strategies based on biochemical markers would not work on previously undiscovered bacteria or on bacteria designed to evade detection. Moreover, genetic sequencing and omics analyses are insufficient to address this growing challenge, since the phenotype of a pathogen might not be determinable from the genetic make-up (i.e., the genotype) alone. The novel capability developed under Friend or Foe will provide detailed, high-throughput phenotype-based characterization of unknown bacteria through identification of pathogenic traits. Specifically, the technology should reliably extract representative samples of bacteria from complex environments, maintain their viability while they are repeatedly interrogated to identify virulence factors, and then analyze them using an omics approach that leverages external pathogen and gene databases. Ultimately, this technology will detect bacterial pathogens as, or even before, they emerge as a threat to the public.

1.2. Technical Approach and Schedule

The performing teams for Friend or Foe will develop complete end-to-end systems that will probe the phenotypes of bacteria to assess their pathogenic potential. The first step will be the extraction and isolation of potentially unknown and/or unculturable bacteria from complex matrices such as laboratory swabs, sewage/run-off, and biofilms. This will be followed by interrogation of the extracted bacteria with a series of non-destructive biochemical and biophysical assays. In the initial phase, interrogation of small populations (<100) of bacteria is acceptable; however, the ultimate goal will be the interrogation of a single bacterium. Bacteria identified as pathogenic should be processed for omics analyses to enable the mapping of phenotype to genotype. For the first two phases of the program, phenotype discovery will be demonstrated on contrived samples composed of known bacteria. The complexity of these samples will increase as the program progresses, ending with real-world samples in Phase III.

These real-world samples will only be examined in conjunction with a Government partner specifically tasked to prevent bacterial outbreaks.

The Friend or Foe program is structured into three Technical Areas (TAs)—Viable Bacterial Isolation (TA1), Interrogation (TA2), and Bioinformatics and Decision Management (TA3)— with an additional and separate Independent Verification and Validation (IV&V) component.

The TAs and IV&V components are described below. As shown in the schematic, the program will progress in three phases. In Phase I the components for each technical area will be tested independently, while in Phase II the components for all TAs must be integrated into a single system that can assess pathogenic phenotypes and then map them to genotypes. In Phase III, the developed technology will be transitioned to a Government partner with whom performers will collaborate to characterize real-world samples. Integrated systems must, at a minimum, meet all characteristics described under individual TAs below, with additional proposal and use case-specific characteristics defined by proposers as needed.

TA1: Viable Bacterial Isolation

TA1 focuses on extracting viable bacteria from their native environments and inserting them into the interrogation platform, while retaining their phenotype for the downstream analysis. This is challenging since in their native environment bacteria are often anchored to surfaces forming complex, heterogeneous communities. Physically separating these bacteria can damage them, while using enzymatic solutions to separate them could potentially degrade surface molecules that are also the virulence factors of interest. Proposals should define high throughput methods that can collect and disaggregate bacteria from complex media and then separate them so that they can be subsequently inserted into the interrogation stations of TA2. Proposals should indicate how the proposed techniques will sufficiently minimize damage to enable subsequent analysis. Preference will be given to technologies that can address complicated samples.

After this initial extraction and isolation, the cells must be maintained at least 24 hours in a state that is sufficiently healthy to respond to the stimuli in TA2. For example, depending on the proposed stimuli in TA2, the cells might need to remain viable through changes in carbon

Phase I Components from each task area will be assessed individually

Phase III Integrated system will characterize real samples with government partner

Phase II Components will be integrated and assessed as a system

TA1 TA2 TA3 TA1 TA2 TA3

Complex, contrived samplesSimple, contrived samples

TA1 TA2 TA3

Real-world samples sources, salinity, temperature, or pH. In general, bacterial activity and viability can depend on the environmental niche (chemical, biological, geological, etc.) where the bacterial community was collected. Proposers should describe how they will characterize a complex medium’s composition and thereby predict the conditions that will maximize cellular viability and preserve natural phenotype. This analysis will guide the selection of media and conditions for extraction and analysis. This approach should also be generalizable to multiple different environmental samples, allowing bacteria to be isolated and interrogated from a variety of sources. Proposals should discuss the necessary resolution of environmental conditions for unbiased sampling, and procedures for adapting techniques to new environments.

The real-world samples examined in Phase III will likely have much greater bacterial diversity than the contrived benchmark samples and will have widely varying population densities.

Proposers should identify the environmental sample that is their primary target as well as two notional alternate samples, providing justification for the selection based on potential pathogenic threat. For the primary target, the proposal should include environmental descriptions that includes projected ranges of bacterial density, structural and biochemical characteristics, and community complexity. It should also include a detailed plan for moving from the contrived samples of Phase II to the real-world samples of Phase III that describes how the full bacterial diversity of the chosen isolate will be addressed. For these complex samples, screening methods to triage bacteria are allowed as long as the full diversity of microbes can still be assayed. While performers must meet the minimum metrics described below in section 1.3, they should also propose a concept of operations (protocol) based on the estimated throughput necessary for success.

Performers must develop methods and devices for (i) isolating viable bacteria from their environments, and (ii) maintaining the cells’ viability and phenotype during interrogation. The minimum requirements for TA1 are as follows:

Methods and devices must isolate individual or small-number consortia of bacteria without destroying viability or damaging cellular features needed for phenotypic characterization;

Media used within the system must maintain viability and enable pathogenic trait interrogation;

Methods and devices must isolate bacteria in numbers sufficient for phenotypic and omics analyses as determined by TA2; and

Methods and devices must accurately capture the full diversity of cells found in the environment being sampled.

TA2: Interrogation

This program aims to develop new analytical techniques that quickly determine the pathogenicity of an unknown bacterium. However, pathogenicity describes a broad range of behaviors, all of which would need to be assessed to determine overall virulence. For this program all proposals must, at a minimum, generate techniques that address the three main categories of pathogenic traits: (1) niche finding (e.g., ability to adhere to host tissues); (2) ability to harm a host (e.g., secretion of exotoxins to damage host cells); and (3) self-preservation (e.g., ability to evade the immune system). Because pathogenic traits are often expressed only in the environment where they are needed—on or near a host cell—experimental assays should work in a medium where this behavior has been activated. Additionally, pathogenicity is often associated with a particular host cell or tissue. Proposers should specify their target cell(s) or tissue and justify their choice.

For the chosen host cell or tissue, the system must characterize pathogenic traits sufficiently to establish the bacteria’s overall virulence. Preference will be given to approaches that provide the fullest possible measure of a bacterium’s virulence. Consequently, multiple assays for each trait class are encouraged. Finally, because the program seeks the capability to identify unknown pathogenic functions or mechanisms, assays should characterize behavior (e.g., ability to bind to a host cell) rather than merely detect the presence of known biomolecular signatures (e.g., presence of a specific bacterial adhesin).

Beyond determining pathogenicity, there are additional challenges inherent in interrogating isolates from environmental samples. First, these isolates may contain only individual or small populations of a bacterium. The small size of bacteria and the low concentrations of associated analytes is at or near the resolution of many detection methods. Consequently, proposals should indicate how the chosen assays have sufficient resolution to reliably identify pathogenic traits from low numbers (5-100) of bacteria. Second, while the clusters of bacteria may contain few bacteria, the total number of bacteria may be quite high. Soil samples, for example, may contain up to 10 billion microbes. Since the program aims to screen samples in less than 24 hours, the proposed techniques must maintain high sensitivity while operating at a sufficient throughput. To attain such throughput, methods must be inherently quick or readily run in parallel. Proposals that rely on assays that are not scalable to the throughput specified in section 1.3, or that cannot ultimately subject individual cells to multiple phenotypic tests, are discouraged.

Finally, the approaches will be constrained by the need for an integrated system. The assays developed in TA2 must integrate with the sampling technology developed in TA1. Consequently, the assays should function with bacterial species with diverse characteristics including size and shape, distinct pathogenic behaviors, and required environmental conditions (carbon sources, pH, trace metals, etc.). Secondly, examining all of the classes of pathogenic traits will likely require a series of nondestructive assays. Proposals should discuss potential interference due to the sequence of assays performed and outline the ability to reconfigure the platform to mitigate challenges. Finally, for integration into the bioinformatics effort in TA3, phenotyped bacteria determined to have high virulence should be available for a final destructive omics-based approach to learn more about their genetics.

Performers must develop quantitative, high-throughput methods and devices for characterizing and assessing the pathogenicity of single bacteria non-destructively with the following characteristics:

Integrated devices/platforms must collectively interrogate the three primary classes of pathogenic traits:

o Niche finding o Ability to harm the host o Self-preservation

Platforms must function accurately with diverse species of bacteria from environments with diverse biochemical and structural compositions; and

For a given tissue or host cell, trait characterization must identify the overall virulence and nature of the threat posed by bacteria.

TA3: Bioinformatics and Decision Management

Algorithms and computational infrastructure that integrate and analyze multiple data types will be needed both to predict pathogenicity and to optimize the sequence of TA2 assays. An additional need for computation comes when a potentially pathogenic bacterium is discovered.

Omics analysis and phenotype-to-genotype mapping should be completed to determine the genes responsible for new pathogenic traits or, at least, provide a molecular barcode of that organism or trait for future identification. Proposals must describe their strategy for performing single-cell omics on likely pathogens. The minimum requirements for TA3 are as follows:

Database infrastructure must integrate data from the pathogen-detection system with external databases, and support analysis of data independent of experimental approach;

Based on pathogenic features identified from the interrogation pipeline, algorithms must automatically sort bacteria for disposal, storage, or omics analysis;

System must perform at least one genomic or transcriptomic analysis on selected bacteria of interest;

System must support rapid processing of omics data for identification of novel or engineered bacteria;

Algorithms must map pathogenic phenotypes to genetic signatures; and Algorithms must identify and direct the system towards the optimal set and order of interrogation assays.

Independent Verification and Validation (IV&V)

Performers addressing the TAs described above will interact throughout the program with an independent verification and validation (IV&V) team, established by DARPA that will help test and validate progress. The IV&V team will consist of subject matter experts from Government, Federally Funded Research and Development Centers (FFRDCs), academia and/or other relevant domains. Over the course of the program, the IV&V team will provide contrived benchmark samples with relevance to the performers’ concept of operations and of increasing complexity so as to allow performers to demonstrate the reproducibility and performance of the developed systems. This increased complexity may result from a larger variety of bacterial species, the inclusion of less well-known or non-bacterial microbes, a wider range of species concentrations, and/or other methods deemed appropriate by the IV&V team in consultation with DARPA. In Phase I, this bacterial population will be mixed with simple simulated media (soil, seawater, etc.). In Phase II, the bacterial population will be mixed with real-world media that has been sterilized.

Analysis of IV&V milestone samples by performer teams should be restricted to assays performed on the platform under development. Bacteria in the IV&V samples will be classified as Risk Group 2 (RG2) or lower according to Appendix B of the National Institute of Health (NIH) Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules.

Inventory of known bacteria included in samples will be provided for regulatory purposes, but contents should be shielded from experimental teams to avoid biasing analytical results.

Proposals must budget and include plans for accepting biological samples from and transmitting analysis results to the IV&V team; and plans should include procedures for firewalling information about sample contents, along with internal and external safety and regulatory filings and protocols.

Samples will be delivered by the IV&V team as described in the milestones and metrics section below. Performer teams will have one (1) week to perform their analyses of sample contents and pathogenic phenotypes and deliver results to the IV&V team for comparison to sample ground truth.

To avoid potential conflicts of interest, performers for HR001118S0025 will not be allowed to compete for the IV&V contract. DARPA is not soliciting proposals for IV&V under

HR001118S0025.

Schedule

The Friend or Foe program consists of three sequential phases of 18, 24, and 6 months, respectively. Progress towards the overall program goal will be assessed throughout the program.

During Phase I, performers will be expected to validate the operation of their prototype systems using a simple contrived sample. By the end of Phase I, the feasibility of integrating components from all three TAs must be demonstrated. By the end of Phase II, performers must demonstrate successful operation of a fully-integrated system on complex contrived samples. Following successful demonstration of the developed technology, DARPA will fund performers to work with Government partners to transition the initial capability to that partner for deployment.

Several potential Government partners will be identified by DARPA. A full system that successfully performs high throughput pathogenic screening analysis on real-world samples shall be delivered by the end of Phase III.

1.3. Program Milestones, Metrics, and Deliverables

Progress toward the program goal will be determined through the use of regular milestones, metrics, and deliverables. The Government specifies the following minimally-required milestones, metrics, and deliverables in order to bound the effort while still affording the maximum flexibility, creativity, and innovation in proposing solutions to the stated problems.

Proposers are expected to define additional quantitative and qualitative success criteria as needed. Proposers must clearly and uniquely itemize tasks needed to accomplish planned milestones and deliverables.

Proposals must be written to address milestones in all three TAs: Viable Isolation (TA1), Interrogation (TA2), and Bioinformatics and Decision Management (TA3). Proposals that do not address all three technical areas will be considered non-conforming and rejected without review. The milestones and metrics for each technical area and phase are outlined below. Proposers must explain quantitative success criteria for each milestone, and information on how it will be achieved, in their Statement of Work (SOW).

Phase I (months 1 through 18)

Technical Area 1: Viable Isolation

Goal: Develop methods for isolating diverse, representative populations of bacteria from samples while maintaining cell viability.

Milestones:

(i) Demonstrate the sorting of 105 bacteria per hour from heterogeneous samples of ≥20 bacterial species from simulated environmental samples (12 months).

(ii) Demonstrate rapid analysis and reproduction of media conditions for unknown simulated environmental samples, with turnaround time of less than 4h from initial analysis to reproduction of conditions (12 months).

(iii)Maintain the viability of isolated bacteria for at least 24 hours (15 months).

(iv)Demonstrate that the isolation system can be integrated with interrogation system (18 months).

In Phase I, performers will develop the technologies necessary to isolate, without significant bias, viable bacteria from complex media. The system must isolate bacteria in sufficient health and abundance to enable subsequent interrogation. The performers should demonstrate that their system can ultimately isolate single bacterial cells by the end of Phase I and that this system can be integrated with the components developed in the other TAs.

The long-term objective of this program is to create a flexible platform and associated protocols for analyzing bacteria from a wide range of environments. With this in mind, the proposed methods should analyze a sample’s composition (pH, nutrients, salts, etc.) with sufficient resolution to create media that mimic this composition and to support the viability of all bacterial community members (supporting viability may not require promoting cell division). Media developed to maintain cell viability should not materially interfere with downstream applications. Methods and devices must be developed with integration across TAs in mind.

IV&V test samples will be provided:

- Month 12, heterogeneous bacterial cultures containing ≥20 species of varied size, shape, motility, and community structure.

- Month 15, heterogeneous bacterial cultures containing ≥20 species mixed into simple, simulated environmental samples.

Metrics:

- More than 50% of bacteria must survive extraction.

- Enough bacteria from each of the initial species must survive to enable analysis by TA2 of all species present in the sample.

- 105 cells extracted and recovered per hour.

Technical Area 2: Interrogation Goal: Develop systems that can characterize the pathogenicity of a small number of bacteria across all three classes of pathogenic traits.

Milestones:

(i) Demonstrate detection of at least one trait for each of the three classes of pathogenic traits

(18 months).

(ii) Test positive and negative control species and deliver data to algorithm development team (18 months).

(iii)Demonstrate feasibility of integration with TA1 and TA2 systems (18 months).

In Phase I, the TA2 objective is to establish the initial set of assays for pathogenicity. The assays must identify at least one pathogenic trait in the three classes defined in this program: niche finding, ability to harm the host, and self-preservation. Though the platform should eventually isolate individual bacteria, such precision is not required in Phase I. Assays sensitive to groups with 100 or fewer bacteria will be sufficient to complete the Phase I TA2 milestone.

Cell viability and phenotype must be maintained throughout testing. This does not mandate that the bacteria continually grow and divide in the assay conditions; however, they should maintain core functions and respond to their environment (i.e., sensing the addition of chemical stimuli or the presence of a host cell). Assays should operate reliably on multiple bacterial species, spanning a range of sizes, shapes, motility mechanisms, and community structures.

Validation will be performed on pure cultures of bacteria provided by the IV&V team. Beyond the samples provided by the IV&V team, proposals should identify a set of positive and negative control strains to be used during development and testing. Data acquired from the TA2 tests during Phase I will be used for the TA3 analysis, providing initial experimental training data for trait identification algorithms.

At the end of Phase I, performers must demonstrate use of the output from the TA1 isolation component as the input to the TA2 interrogation system. Performers must also demonstrate that the output from the interrogation system can be used with the single-cell omics analysis from

TA3.

The TA2 objectives do not require operation at the final throughput and resolution required for this program by the conclusion of Phase I. However, a detailed plan for scaling the analysis platform to the final target performance should be provided to DARPA by the conclusion of Phase I. Key metrics of success include the time needed for discrimination of each cell or cluster of cells, the number of traits distinguished, and the number of cells (or clusters of cells) that can be analyzed per run.

IV&V test samples will be provided:

- Month 12, ≥20 different bacterial strains with varied pathogenic traits, from pure cultures.

Metrics:

- Detectors must be sensitive enough to detect pathogenic traits at the level of ≤100 bacteria.

- Platform must function with all types of bacteria provided by the IV&V team (approximately

20 species at Month 12).

- Viability after testing across all three pathogenic trait classes, as determined by viability stains or other measure of metabolic activity.

Technical Area 3: Bioinformatics and Decision Management

Goal: Demonstrate successful pathogenic trait prediction using defined cultures and simple manufactured samples.

Milestones:

(i) Detailed plan for integration of the components from each TA (15 months).

(ii) Methods that fuse phenotype and single-cell omics data, to support mapping of detected pathogenic characteristics to genetic traits (18 months).

(iii)Training data set constructed using control samples and external data, of sufficient size and detail for use in pathogen identification (18 months).

(iv)Algorithms for identifying bacteria of interest based on pathogen assays and omics data (18 months).

(v) Demonstrate ability to integrate single-cell omics system with interrogation platform (18 months).

Metric: Predictive pathogenic trait identification with ≥90% accuracy on control samples.

In Phase I, performers will develop a computational and experimental framework to support all data analysis tasks associated with their effort. The framework should enable data manipulation that fuses multiple different data types, maps genotype-to-phenotype, and identifies potential pathogens. Computational frameworks that readily extend to incorporate new types of assays and data beyond those in the performer’s specific effort are welcome.

Performers will provide a storage solution for data generated by TA2, as well as any additional external data that may support the effort (e.g., phenotype data extracted from existing literature or provided by other performers). Data storage solutions that can be easily transitioned so that they “live on” beyond the lifespan of the Friend or Foe are preferred.

Performers will develop and implement methods for fusion of multiple data types and for identifying bacteria of interest based on the combined data. Performers must also construct an appropriate training data set using control samples developed by TA2 along with external data as needed. Performers are encouraged to incorporate as much data as possible relevant to pathogenicity and bacterial identification. The scope of this work may include methods for large-scale mining and processing of existing literature. The sufficiency of the training data and validity of any algorithms must be confirmed by the end of Phase I using samples provided by the IV&V team; performers must demonstrate pathogen identification with ≥90% accuracy on the IV&V samples described under TA1 and TA2 by the end of the phase.

Performers must implement single-cell omics techniques to complement the phenotypic assays and to generate genotype data for use in phenotype-to-genotype mapping. Performers may adapt existing methods and/or develop new methods as appropriate. As with the phenotypic data, omics data must be stored and manageable within the developed computational framework.

Phase II (months 19 through 42)

Technical Area 1: Viable Isolation

Goal: Integrated system that isolates diverse, representative populations of bacteria from increasingly complex samples while maintaining cell viability.

At the end of Phase II, performers must provide a mitigation strategy dealing with any remaining challenges their technology will face in dealing with real-world samples. In particular, they will need to deal with the greater biological and chemical diversity of such samples. To manage the biological diversity, the isolation strategy in Phase II may include triage to rapidly separate the cells of greatest interest to researchers, provided that it does not reduce the overall accuracy below the limits set in TA2. The strategy should also address any substances that occur naturally in their targeted environmental samples (e.g., metals, pollen, humic acids, detergents, etc.) that may confound their detection approaches.

Milestones:

(i) Must isolate bacteria from communities of 50-100 cell types from:

o Samples consisting of a complex, contrived environmental samples (30 months) o Sterilized real-world media (soil, seawater, etc.) (36 months).

(ii) Operate as an integrated unit with interrogation platform from TA2 (21 months).

(iii)Detailed transition plan that addresses analysis of real-world samples (36 months).

The Phase II goals for TA1 are focused on extending the technology developed in Phase I to operate on increasingly complex samples, with 10-fold higher throughput per unit time. IV&V samples must be processed as an integrated platform with TA2.

By the end of Phase II, the performers must demonstrate that their integrated system is able to process the increasingly complex samples provided by IV&V, with reliable identification of members of the communities with pathogenic traits. More than 80% of initial bacteria must survive the extraction process, while maintaining the relative abundance of community members.

At least some representatives of each cell type must survive, and throughput must be increased to 106 cells per hour.

IV&V test samples will be provided:

- Month 19, contrived samples with communities of 50-100 different cell types mixed into a simple medium.

- Month 30, contrived samples with bacteria of 50-100 different cell types mixed into a more complex medium. Here, complex means more potential confounding agents such as metabolites, metal ions, or mineral sources.

- Month 36, quasi-environmental samples consisting of 50-100 different cell types mixed into sterilized real-world samples.

Metrics:

- More than 80% of bacteria must survive extraction.

- Enough bacteria from each of the initial species must survive to enable analysis by TA2.

- 106 cells extracted and recovered per hour.

Technical Area 2: Interrogation

Goal: Develop an integrated system that successfully performs high throughput pathogenic screening analysis on increasingly complex samples. The number of tests should be sufficient to establish the pathogenicity of the bacteria for the chosen host cell or tissue.

Milestones:

(i) Operate using output from integrated isolation platform from TA1 (month 36).

(ii) Operate as integrated unit with single-cell omics platform from TA3 (month 36).

(iii)Improve the sensitivity of detectors to operate at the level of single bacteria (month 42).

(iv)Within the context of a chosen host cell or tissue, perform ≥2 assays for each of the three classes of pathogenic traits or otherwise perform a sufficient number of assays to identify 90% of the pathogenic strains in the IV&V samples in the context of a chosen host cell or tissue (month 42).

During Phase II, TA2 will expand the technology developed in Phase I to work on increasingly complex samples, at throughput compatible with intended use cases. Assays will be refined to operate at resolution compatible with single cells, from the previous constraint of ≤100 bacteria.

By the end of Phase II, the TA2 assays must be able to map pathogenic traits to single bacteria, in a platform integrated with the viable isolation process developed in TA1. These assays must also work with the technologies in TA1 and TA3 to form a fully-integrated platform ready for transitioning to a Government partner in Phase III.

Metrics will include the time needed to analyze each input sample, the number of traits distinguished, and the number of cells (or clusters of cells) analyzed per run.

IV&V test samples:

Since the system will be integrated at the beginning of this phase, the IV&V samples are the same as for TA1.

Metrics:

- Detectors must be sensitive enough to analyze single bacteria.

- Platform must function with all types of bacteria provided by the IV&V team.

- A sufficient variety of traits must be assessed to establish the pathogenicity of the bacterium for the chosen host cell or tissue.

Technical Area 3: Bioinformatics and Decision Management Goal: Demonstrate successful pathogenic trait detection using increasingly complex contrived samples and an efficient interrogation pipeline, and assign genetic origins of the identified pathogen-associated phenotypes.

Milestones:

(i) Optimized predictive threat identification algorithms with improved performance over Phase

I (month 36).

(ii) Algorithms for decision tree optimization (month 36).

(iii)Algorithms for mapping phenotype-to-genotype (month 36).

NOTE: Proposers should provide interim milestones to demonstrate progress against the objectives listed above.

Metrics:

(i) Predictive pathogenic trait identification with ≥95% accuracy on control samples.

(ii) Increased pipeline efficiency with reduction (over Phase I) in number of assays required for successful pathogenic trait identification.

(iii)≥90% accuracy in mapping of pathogen phenotypes to known genetic features in control sample species.

Computational and analytic methods developed in Phase I shall be extended and improved in Phase II to enable improved prediction accuracy (≥95%) on the more complex samples provided by the IV&V team. Accuracy includes correct identification of bacteria with and without known pathogenic traits.

Performers must develop algorithms for the interrogation pipeline that optimize the assay decision tree. Reports should detail precisely how these algorithms represent an improvement over the baseline decision tree algorithm used in Phase I or some other nominal algorithm.

Performers shall demonstrate the efficiency improvement by characterizing the interrogation approach (e.g., by time to positive identification, number of assays) and contrasting with the process from Phase I.

Performers must demonstrate successful mapping of phenotype-to-genotype for identified bacteria with pathogenic traits, by identifying the specific genetic sequences associated with those characteristics. Genotype must be established using the single-cell omic tools developed in Phase I (and extended in Phase II, as needed).

Data collected during Phase II must fit into the larger computational framework of the effort.

Control data from known samples may be added to the training dataset created in Phase I.

Phase III (months 43 through 48)

Phase III focuses on transitioning to a Government partner a high-throughput system that can identify pathogens in real-world samples. Performers that have demonstrated a system with sufficient throughput and accuracy will be funded to transfer that system to a Government partner with whom they will collaboratively examine a real-world sample and so establish the duplicated system’s performance.

Technical Area 1: Viable Isolation Milestone: Protocol that successfully performs bacterial isolation from high-value real-world samples (month 45).

The key metrics for TA1 in Phase III will be similar to the Phase II metrics. The performers will aid the Government transition partner with duplication of a fully-integrated, high-performing system with detailed protocols and instructions.

The performers and Government transition partner must show that the transferred platform shows comparable throughput and sensitivity when operating on high-value samples. This will include statistics on time taken to analyze each sample, number of traits distinguished, and accuracy in reflecting the composition of the input community.

Technical Area 2: Interrogation Milestone: Integrated analysis done in replicated system with Government partner that detects pathogenic traits of bacteria in natural samples (month 48).

The Phase III TA2 goals are to ensure that the assays function at the resolution and throughput needed for platform transfer to a Government partner. The proposer will collaborate with the Government transition partner to ensure that the assays work at the reliability and precision needed in the transferred platform.

Assays in Phase III must work at resolution compatible with single cells, on realistic samples relevant to the applications of the platform in the real-world. Validation will be confirmed upon transfer to the Government partner and must reflect the known composition of real-world samples that contain pathogens.

Technical Area 3: Bioinformatics and Decision Management Milestone: Transfer of fully-documented data analysis platform to Government partner (month 45).

The Phase III TA3 goal is to ensure the successful transfer of the data-processing and decision system to the Government transition partner. This will include transfer of fully-documented software, as well as any relevant databases based on both publicly-available databases and any new training data collected in the course of this program.

The key metric of success in this phase will be IV&V verification of the performance of the bioinformatics and decision management system on the samples identified by the Government partner and drawn from the environment or clinical settings.

1.4. General Requirements

Proposing Teams

Proposer teams must address all three technical TAs described above which should run in parallel. Consequently, it is expected that the teams will include experts from the multiple disciplines related to the program challenges and goal (e.g., bacterial pathogenesis, single-cell assay development, microfluidics, metabolomics/genomics/proteomics, bioinformatics). Because several different technologies must ultimately work together, teams are encouraged to identify one or more members as project integrators who will ensure that team members focused on a specific TA are also appropriately working towards the overall program goal. The project integrator should also address all risks specifically associated with integration.

Specific content, communications, networking, and team formation are the sole responsibility of the proposer teams. Proposer teams must submit a single, integrated proposal led by a single Principal Investigator or prime contractor.

DARPA will hold a Proposers Day (see Section 8, Other Information) to help facilitate the formation of proposer teams and to enable sharing of information among interested parties through the DARPA Opportunities Page and the Proposers Day registration website.

Data Sharing

DARPA anticipates that a large amount of data will be generated under this program by each performer. DARPA encourages sharing of data generated in the course of this program, as well as data aggregated for the integrated analysis workflow from pre-existing work or research funded by other sources, although this is not a requirement of the program.

Biocontainment and Biosafety

As the core of the program requires characterization of environmental samples that may include live pathogens and control samples consisting of known pathogens, the Friend or Foe program will be conducted in appropriate biocontainment facilities in accordance with Center for Disease Control (CDC) regulations. Friend or Foe will not support any proposals that include uncontained experiments and/or environmental release of pathogens. DARPA follows all regulations and laws related to pathogen detection.

The Friend or Foe program is structured such that Phase III is the only time at which performers will use their integrated platform to process real-world samples with known or anticipated pathogenic bacteria. In the event that a performer wishes to use their platform to analyze real-world samples potentially containing new or known bacterial species that may qualify as Select Agents, DARPA requires that the performer must notify DARPA of this intention in advance.

This notification must include information on an appropriate Government agency partner (e.g., Defense Threat Reduction Agency (DTRA), United States Army Research Institute for Environmental Medicine (USARIEM), Armed Forces Health Surveillance Center (AFHSC), Center for Disease Control (CDC), Department of Homeland Security (DHS), etc.) that has agreed to work with the performer’s team to ensure compliance with CDC select agent regulations. It must also contain a detailed justification and explanation of the performer’s intended experiments, including proposed containment strategies to prevent unauthorized access, theft, loss, or environmental release of potential select agents. Any such proposals are subject to DARPA approval. DARPA will not support any research on real-world samples that is not supervised by an appropriate Government partner organization.

Technology Transfer

Proposals must discuss the transition of the technology developed to a Government partner, including plans for partner engagement, IP issues, logistics of reproducing the technology, and any non-commercial items required. DARPA will identify a set of potential partners for technology transfer; however, performers may identify other Government partners that can be pursued at DARPA’s discretion. Notional needs and examples of clinical or environmental samples will be presented at the Proposer’s Day, slides from which will be available at http://www.darpa.mil/work-with-us/opportunities. Proposals must budget resources for the development and execution of a Technology Transfer Plan that describes steps for a successful engagement and transfer of data and methods. A formal plan must be finalized by six months prior to the conclusion of Phase II, with transition to Phase III requiring active engagement with a Government partner. If awarded, DARPA must be included in the development of potential technology transfer relationships.

Other Requirements

Performers are expected to attend semi-annual program reviews to provide updates to the DARPA program management team and other Friend or Foe performers on progress towards their milestones and scientific goals on the Friend or Foe program. Performers will also summarize outstanding challenges and limitations that must still be overcome to achieve the overarching goals of the program.

2. Award Information

2.1. General Award Information

Multiple awards are possible. The amount of resources made available under this BAA will depend on the quality of the proposals received and the availability of funds.

The Government reserves the right to select for negotiation all, some, one, or none of the proposals received in response to this solicitation and to make awards without discussions with proposers. The Government also reserves the right to conduct discussions if it is later determined to be necessary. If warranted, portions of resulting awards may be segregated into pre-priced options. Additionally, DARPA reserves the right to accept proposals in their entirety or to select only portions of proposals for award. In the event that DARPA desires to award only portions of a proposal, negotiations may be opened with that proposer. The Government reserves the right to fund proposals in phases with options for continued work, as applicable.

The Government reserves the right to request any additional, necessary documentation once it makes the award instrument determination. Such additional information may include but is not limited to Representations and Certifications (see Section VI.B.2., “Representations and Certifications”). The Government reserves the right to remove proposers from award consideration should the parties fail to reach agreement on award terms, conditions, and/or cost/price within a reasonable time, and the proposer fails to timely provide requested additional information. Proposals identified for negotiation may result in a procurement contract, cooperative agreement, or other transaction, depending upon the nature of the work proposed, the required degree of interaction between parties, whether or not the research is classified as Fundamental Research, and other factors.

Proposers looking for innovative, commercial-like contractual arrangements are encouraged to consider requesting Other Transactions. To understand the flexibility and options associated http://www.darpa.mil/work-with-us/opportunities with Other Transactions, consult http://www.darpa.mil/work-with-us/contract-management#OtherTransactions.

In all cases, the Government contracting officer shall have sole discretion to select award instrument type, regardless of instrument type proposed, and to negotiate all instrument terms and conditions with selectees. DARPA will apply publication or other restrictions, as necessary, if it determines that the research resulting from the proposed effort will present a high likelihood of disclosing performance characteristics of military systems or manufacturing technologies that are unique and critical to defense. Any award resulting from such a determination will include a requirement for DARPA permission before publishing any information or results on the program. For more information on publication restrictions, see the section below on Fundamental Research.

2.2. Fundamental Research

It is DoD policy that the publication of products of fundamental research will remain unrestricted to the maximum extent possible. National Security Decision Directive (NSDD) 189 defines fundamental research as follows:

‘Fundamental research’ means basic and applied research in science and engineering, the results of which ordinarily are published and shared broadly within the scientific community, as distinguished from proprietary research and from industrial development, design, production, and product utilization, the results of which ordinarily are restricted for proprietary or national security reasons.

As of the date of publication of this BAA, the Government expects that program goals as described herein may be met by proposers intending to perform fundamental research and does not anticipate applying publication restrictions of any kind to individual awards for fundamental research that may result from this BAA. Notwithstanding this statement of expectation, the Government is not prohibited from considering and selecting research proposals that, while perhaps not qualifying as fundamental research under the foregoing definition, still meet the BAA criteria for submissions. If proposals are selected for award that offer other than a fundamental research solution, the Government will either work with the proposer to modify the proposed statement of work to bring the research back into line with fundamental research or else the proposer will agree to restrictions in order to receive an award.

Proposers should indicate in their proposal whether they believe the scope of the research included in their proposal is fundamental or not. While proposers should clearly explain the intended results of their research, the Government shall have sole discretion to select award instrument type and to negotiate all instrument terms and conditions with selectees. Appropriate clauses will be included in resultant awards for non-fundamental research to prescribe publication requirements and other restrictions, as appropriate. This clause can be found at http://www.darpa.mil/work-with-us/additional-baa.

For certain research projects, it may be possible that although the research being performed by the awardee is restricted research, a subawardee may be conducting fundamental research.