G

Nucleic-Acid Signatures at Three Levels of
Readiness for BioWatch

R. Mariella, Jr., Ph.D.

A white paper prepared for the June 25–26, 2013, workshop on Strategies for Cost-Effective and Flexible Biodetection Systems That Ensure Timely and Accurate Information for Public Health Officials, hosted by the Institute of Medicine’s Board on Health Sciences Policy and the National Research Council’s Board on Life Sciences. The author is responsible for the content of this article, which does not necessarily represent the views of the Institute of Medicine or the National Research Council.

The paper by Raymond P. Mariella, Jr., will discuss nucleic-acid signatures (polymerase chain reaction [PCR], microarrays, and other probe-based systems) at three levels of readiness for BioWatch:

•   Tier 1: fully automated biodetection system, capable of 24/7/365 unattended outdoor and indoor operation, that will be at a technology readiness level of technology readiness level (TRL) 6-plus by 2016.

•   Tier 2: similar requirements but will not reach a TRL 6-plus level until sometime between 2016 and 2020.

•   Tier 3: technologies that have the potential of meeting or exceeding the BioWatch requirements, but a fully automated, TRL 6-plus system would take us beyond the 2020 time frame. For these technologies, describe the current critical paths (“long poles”) in meeting the BioWatch requirements and how they might be addressed.



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G Nucleic-Acid Signatures at Three Levels of Readiness for BioWatch R. Mariella, Jr., Ph.D. A white paper prepared for the June 25–26, 2013, workshop on Strate- gies for Cost-Effective and Flexible Biodetection Systems That Ensure Timely and Accurate Information for Public Health Officials, hosted by the Institute of Medicine’s Board on Health Sciences Policy and the Na- tional Research Council’s Board on Life Sciences. The author is respon- sible for the content of this article, which does not necessarily represent the views of the Institute of Medicine or the National Research Council. The paper by Raymond P. Mariella, Jr., will discuss nucleic-acid sig- natures (polymerase chain reaction [PCR], microarrays, and other probe- based systems) at three levels of readiness for BioWatch:  Tier 1: fully automated biodetection system, capable of 24/7/365 unattended outdoor and indoor operation, that will be at a tech- nology readiness level of technology readiness level (TRL) 6- plus by 2016.  Tier 2: similar requirements but will not reach a TRL 6-plus lev- el until sometime between 2016 and 2020.  Tier 3: technologies that have the potential of meeting or exceed- ing the BioWatch requirements, but a fully automated, TRL 6- plus system would take us beyond the 2020 time frame. For the- se technologies, describe the current critical paths (“long poles”) in meeting the BioWatch requirements and how they might be addressed. 155

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156 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH DISCLAIMER This paper, focusing on nucleic acid–based detection of bioaerosol agents, must be only a subset of a larger plan. This paper does not ad- dress foodborne, waterborne, vectorborne, or human-to-human transmis- sion of infectious agents. AUTHOR’S STRONG RECOMMENDATION FOR BOTH PLANNING AND IMPLEMENTATION Because the committee is examining what would be entailed to pro- vide an autonomous system that performed BioWatch assays and provid- ed the Laboratory Response Network (LRN) of the Centers for Disease Control and Prevention (CDC) with the same amount of nucleic-acid sequence information, along with the same reliability and accuracy as the validated laboratory assays, in the author’s opinion the CDC/LRN and any others who will rely upon such information to make calls of detec- tion of an agent need to be involved in each step in the planning and de- velopment of such an autonomous system. CAVEAT: FULL, END-TO-END SYSTEM INTEGRATION IS DIFFICULT! The author’s general premise is that the integration of disparate components into an automated bioagent detection and identification sys- tem that attempts to perform the same functions as trained laboratory personnel is extraordinarily difficult. Numerous high-quality commercial off-the-shelf instruments exist that perform the functions of the individu- al steps, yet field-tested and proven integrated systems are few. It is a particular challenge in any automated fluidic system to make 100 percent reliable fluidic interconnections along with the necessary methods to transport, meter, and process samples and reagents so that the laboratory- proven assays that normally are carried out by trained personnel with pipettes, filters, and centrifuges perform just as well in the automated system. Challenges to system integration can arise from something as basic as an output volume of one stage being incompatible with the next stage’s input volume or from differing buffer concentrations between one stage and the next or from a “short spin” on the centrifuge being needed

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APPENDIX G 157 in between, and so on. One extremely important hurdle for system inte- gration is that a manufacturer may refuse to provide what seems to be a minor modification to a selected component that is needed to permit in- tegration, and, if a third party performed the needed modification, the original manufacturer may refuse to service the modified component and declare that the modification voided the warranty. This is particularly likely when the desired component might be projected to have total an- nual sales of a few dozen or less. Thus, a stand-alone instrument may work very well in an LRN or BioWatch laboratory and, yet, effectively not be available for system integration. INTRODUCTORY THOUGHTS AND OVERVIEW In Figure G-1, items 2 through 4 and 6 through 11 are either incorpo- rated or could easily be incorporated in systems that could be deployed by 2016. Item 1, precollection particle characterizer, could probably be incorporated as part of a next-generation system by 2016, but certainly by 2020, if the cost-benefit analysis favored its incorporation. Item 5, selective capture/enrichment of pathogens via surface molecular recogni- tion is listed, although extensive research and development would be re- quired to make this a reality (Pratt et al., 2011). Item 8 is also somewhat of a catchall, covering simple chemistries, such as the Boom cap- ture/release of nucleic acids, but may also include emerging techniques, including even capture, isolation, and release of nucleic-acid regions of interest on either fixed or liquid microarrays (DuBose et al., 2013). Item 12 would likely require its own multiplex target amplification, prior to hybridization, and incorporating such a process, along with the logistics of replacement of single-use microarrays or the reconditioning of a mi- croarray, along with automated readout of hybridization patterns, seems destined for deployment beyond 2020. Item 13 would perform a very desirable function, but attempting an automated metagenomic analysis of a sample seems like a very “long pole” in the tent. Preceding sequencing with multiplex target amplification may make this automatable by 2020.

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158 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH PERFORMANCE CONSIDERATIONS Premise A release of agent can dissipate and yet remain at physiologically relevant concentrations, for large areas, putting the public at risk, so the aerosol sampler needs to bring in as many liters per minute as is practical. Considerations Without regard to any specific autonomous system or placement of a multitude of such systems throughout an urban setting, let us simply con- sider what might happen to persons who were exposed for 30 minutes to a release that contained N viable bacteria/m3. From Chapter 6 of the Environmental Protection Agency (EPA) Ex- posure Factors Handbook (Moya et al., 2011), adult humans respire, on a 24-hour average, roughly 10 liters/minute, so 30 minutes = 300 liters breathed. Because 1,000 L= 1m3, “average” breathing would pull in 0.3 N viable bacteria over 30 minutes. Depending upon the details of the aer- osol, it could happen that effectively every viable bacterium breathed in could be retained within the lungs. The infectious dose of some bacteria and viruses has been measured to be essentially a single viable bacterium or particle with infectious viri- ons (Jones et al., 2005). Regarding viruses, according to Kamps (2006): Within nasal secretions, millions of virus particles per ml are shed, so that a 0.1–µ1 aerosol particle contains more than 100 virus particles. A single HID (human infectious) dose of influenza virus might be be- tween 100 and 1,000 particles. (p. 91) Although respiration rates among the very young or the elderly may be lower, the infectious dose for some agents may be lower as well. Accord- ing to Ingles by et al. (2002): Recently published extrapolations from primate data suggest that as few as 1 to 3 spores may be sufficient to cause infection (Peters and Hartley, 2002). The dose of spores that caused infection in any of the 11 patients with inhalational anthrax in 2001 could not be estimated although the 2 cases of fatal inhalational anthrax in New York City and Connecticut provoked speculation that the fatal dose, at least in some individuals, may be quite low. (p. 2239)

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FIGURE G-1 Block diagram of components of a notional next-generation system, including compo- 159 nents and functions that may be incorporated in a staged manner.

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160 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH Thus, if N for a particular agent remained above 10/m3, for example, then a 30-minute exposure could lead to infection of persons in that vi- cinity if the infectious dose was very low. The location of an autonomous system may be far enough away that the aerosol concentration of poten- tially pathogenic bacteria or virus may have dropped to a much lower level when the release reached the detection system; indeed, this appen- dix author argues that the farther apart one wishes to locate such auton- omous systems, the higher each system’s volumetric sampling and sensitivity need to be to avoid missing an attack. Regarding background bioaerosols, Jensen (2007) reported: Time-resolved concentration profiles of bacteria and spores showed that spore concentrations of 11,000 per cubic meter occurred and that the concentration curve contained many “bumps” resembling an aero- sol cloud passage as well as many short duration spikes. (p. 9) Jensen added that “there were approximately 50 total airborne bacteria for every culturable bacterium” (p. 9), which is of relevance for assays, such as PCR and most other nucleic acid–sequence-based assays, that do not distinguish between “live” and “dead” bacteria. All of this is to say that it is not hard to envision a situation in which a potentially pathogenic bacteria or virus could be present at physiologi- cally relevant concentrations, while remaining well below the back- ground concentration of physically similar, nonpathogenic aerosolized bacteria and viruses. NUCLEIC-ACID SIGNATURES What comprises nucleic-acid signatures? Should an autonomous sys- tem include genomic, plasmid, and even rRNA in bacteria by 2020? Concerning informatics, what level of sequence ID is required to call a positive? How well conserved are “consensus sequences”? How much deviation is acceptable? (This is an especially tricky issue for single- stranded DNA and RNA viruses.) Experimentally, what level of se- quence mismatch or incorrect base calling is acceptable? How do signatures need to change as strains evolve or as new spe- cies and subspecies are discovered (Jackson et al., 2012; Signarovitz et al., 2012; Zeytun et al., 2012), both within the species of the pathogen and with its near neighbors?

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APPENDIX G 161 The author expects that such discussions of nucleic-acid signatures and their evolution over time will proceed within the workshop panels “BioWatch—Information for Decision Making” and “State of the Art on Genomic Sequencing.” However, this author wishes to convey, anecdo- tally, that it is his understanding that for at least one species of pathogen- ic virus there are no conserved sequences longer than 14 bases, making a single-target assay problematic for that agent. Moreover, for single- stranded DNA and RNA viruses, concepts such as “quasispecies” are often needed and appropriate (Lauring and Andino, 2010; Volz et al., 2013). ASSAYS LRN and BioWatch laboratory assays today are certainly performed with commercial instruments, but due to the considerations at the begin- ning of this report on the challenges of system integration, we will not specifically compare various commercial, stand-alone components or instruments. However, the author hopes what follows will be discussion provoking. Digital Sample Processing and Digital PCR Given the rigor and time-consuming nature of creating fully tested and validated assays within the guidance of public health actionable as- says,1 digital PCR is likely not fieldable by 2016, but could possibly be TRL 6 by 2020 (Vogelstein and Kinzler, 1999). 1 See “Framework for a Biothreat Field Response Mission Capability,” developed by an interagency working group convened to develop guidance to first responders for the biologi- cal assessment of suspicious powders. Public health actionable assays (PHAAs) are laboratory-based assays that are used to sup- port public health decisions and which have been qualified according to consensus perfor- mance standards developed by a recognized and representative body from the stakeholder community. PHAAs are developed and utilized to support public health actions involving the potential exposure of an individual or, more commonly, groups of individuals to biothreat materials such as Ba spores. PHAAs have high specificity, high sensitivity, and are highly robust to provide critical information on agent-specific confirmation and further characteriza- tion to support public health decisions such as initiating a national or local health alert warn- ing, initiating a public health investigation, conducting risk assessments to support post- exposure prophylaxis distribution, and initiating public health risk communications. These assays are intended to be employed in well-established controlled laboratory environments,

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162 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH Gevensleben and colleagues (2013) detected and quantified circulat- ing free plasma DNA with droplet-digital PCR (ddPCR); Strain et al. (2013) did HIV quantification as did Henrich et al. (2012). Kelley et al. (2013) reported sensitive quantification of methicillin-resistant Staphylo- coccus aureus using digital PCR. Roberts and colleagues (2013) de- scribed a digital PCR diagnostic assay for ocular Chlamydia trachomatis; unlike other nucleic-acid amplification tests, it “requires no external or internal calibration yet delivers a highly accurate estimation of target load.” Speaking about quantification, Morisset and colleagues (2013) wrote: The excellent performance of the tested parameters enables the quanti- fication of samples from different matrices, using DNA extracted with common methods without up-front DNA quantity estimation. The lim- its of quantification, trueness, and repeatability of the duplex assay comply with international recommendations. They also noted that “ddPCR [droplet digital polymerase chain reaction] running costs are lower than those of standard qPCR [quantitative poly- merase chain reaction] technology, given the superior throughput.” There are numerous other publications that used a variety of plat- forms (Ottesen et al., 2006; Shen et al., 2011; Straub et al., 2013; Tadmor et al., 2011; Whale et al., 2012). Exactly how a system integrator would implement digital PCR, even by 2020, is an open question, but worth considering. An Aside on Limits of Detection2 Apropos of the previous discussion of “digital PCR,” but without diving into what is famously known as “receiver operating characteris- tic” curves, let us briefly review what is meant by a “false-negative.” It has been demonstrated, for almost as long as PCR has been used, that an optimized PCR assay can perform down to the Poisson limit of detection such as an LRN reference laboratory using an established ConOps and where professional training and user proficiency certification are established. 2 This author understands that a careful study was made of BioWatch assays, implemented under both single-plex and multiplex formats, but as of this writing he has not been able to read any resulting report. Such a study would bear upon the discussion about limits of detec- tion and Poisson statistics in the preceding section.

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APPENDIX G 163 (Sykes et al., 1992). That is, if the PCR reaction solution has no inhibi- tors but does have the appropriate primers, master mix, and only a single copy of the target DNA, a positive detection signal can be observed (Sykes et al., 1992). Without referring to any particular autonomous in- strument, if it collects 1 m3/min and has a 10 percent overall processing throughput from collection to PCR reaction, including archiving and di- viding some of the collected sample, then if a particular bacterium enters the collector as an aerosol with concentration of 10/m3 and the system collects air for 1 minute, then on average the PCR reaction will have one bacteria target in its reaction volume. However, assuming that Poisson statistics apply, 1/e = 37 percent of the time there will be no target in the PCR reaction, and one can, therefore, expect to “miss” the detection of this aerosolized bacteria at this aerosol concentration 37 percent of the time. By contrast, if there were, say, three separate PCR chambers, each with 10 percent overall throughput from collector to it, each with a dif- ferent sequence signature from the bacterium, then the bacterium would miss all three reactions only 1/e3 = 5 percent of the time. If the manufac- turer claimed a limit of detection with all three signatures positive for this bacterium of 10/m3 in 1 minute, then the system would fail to give this positive 37 percent of the time. If only one or two signatures came up positive, the result could be termed “indeterminate,” for example, with its own particular concept of operation. Also note that if a multiplex assay with three sequence signatures per target organism also obeyed Poisson statistics and was incorporated in a similar autonomous system that had 30 percent throughput from collector to reaction chamber, because of having less division of the collected sample, it would produce a false-negative 5 percent of the time for this concentration of aerosol. Topics for Probe-Based Assays  What are the accuracy, time to perform, cost . . .?  Sensitivity, specificity: Should require positive call for all or only some physiologically relevant concentrations? No false- positives from nonvirulent subspecies.  Time to perform assay (Maltezos et al., 2010): “Using Thermo- coccus kodakaraensis polymerase . . . we obtained an accurate, 35-cycle amplification of an 85-base pair fragment of E. coli O157:H7 Shiga toxin gene in as little as 94.1 s.” It seems unlikely that the time to perform the polymerase-based amplification will

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164 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH be the limiting factor on processing time, even with slower ther- mal cyclers.  Difficulty to perform manually and/or automatically.  What level of sample preparation is required?  Ability to transfer informatics, reagents to other locations.  Cost of startup for each detection system, consumables, instru- ment maintenance.  Ability to include positive and negative controls.  Expandability: multiplex versus single-plex.  What protocol and procedures are required for validation (PHAA)? Time, number of assays to run, experimental accuracy, etc. Should there be at least two simultaneous or sequential hybridization events required per signature region, with at least three signatures re- quired per target bacterium or virus? This author, in contacting numerous practitioners, has found diver- gent positions on the topic of which assays to incorporate into autono- mous detection systems. A number of skilled laboratorians endorse the BioPlex® assay, based on multiplex PCR amplification and subsequent hybridization to a liquid array of bead types. A different group of skilled laboratorians endorses real-time PCR with Taqman® or Molecular Bea- con® probes. The BioPlex® assay was fielded for the Department of Homeland Security (DHS) in the Northrop Grumman autonomous path- ogen detector system, with seemingly no false-positives or false-nega- tives. The real-time PCR was tested in the Hamilton Sundstrand M-BAND in realistic settings with good results. Given the rushed nature of the preparation of this report and the relative lack of overwhelming data, no specific recommendation is made in this report, other than to recommend detailing the time and trouble, within the PHAA guidelines (see footnote 2), to produce and validate each approach, both as individual signatures evolve and as the number of target bacteria and viruses increases. Will positive and negative controls be required for each reaction volume? Role of Viability/Infectivity Assays? Part of a long-term system design could include local viability as- says, triggered by initial positive identification with nucleic-acid signa- ture. Rapid viability assays have been developed that can even be applied to slow-germinating Bacillus endospores (Kane et al., 2006; Letant et al.,

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APPENDIX G 165 2010, 2011), but using them would require profound changes in automa- tion, because actual culture is required. For some endospores, an acceler- ated method can still require 12 to 24 hours for accurate results (Letant et al., 2011). Incorporation would require an analysis of the trade-offs between dispatching a courier to retrieve a positive sample for viability and other testing at a staffed laboratory versus attempting to incorporate such an assay in a stand-alone system or some combination of both. Sequencing by Hybridization to Planar Array The author sees sequencing by hybridization (Drmanac et al., 1989; Palacios et al., 2007; Thissen et al., 2010) as having major obstacles to incorporation into a stand-alone, autonomous system, even by 2020. Topics such as probe length and the stringency of hybridization specifici- ty, informatics and pattern-recognition imaging equipment and soft- ware, and trade-offs between copy number, size of array, and time to equilibrate can probably be addressed by 2020, but can sample prepara- tion be automated, and what sort of consumables supply and robotic han- dling of chips for performing assays would be needed? Would the system reuse chips or sequentially use minichips on a larger chip with compli- cated fluidics? What accuracy results from hybridizing with a complex environmental mixture? To what extent could selective multiplex ampli- fication of regions of interest knock down confounding sequences and nonspecific binding? To this author, the “long poles” for this application are establishing and automating the sample preparation that is needed prior to hybridiza- tion as well as robotic chip handling and the necessary fluidics for intro- ducing new chips and enabling equilibrated hybridizations. The overall cost versus performance would also need to be considered. Direct Sequencing of Probe Regions Direct sequencing of probe regions can be done via pyrosequencing, single-molecule sequencing, etc. Although next-generation sequencing continues to increase the power and affordability of sequencing a hu- man’s genome, how well do these increases enhance rapid, autonomous sequencing of pathogenic bacteria and viruses from complex environ- mental samples?

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166 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH There are a variety of issues concerning sample preparation for direct sequencing. Starting from a raw aerosol sample—such as that captured in a wetted-wall particle collector, for example—ideally a fully automated system should be able to perform the following tasks:  Possible selective capture of virulent bacteria and viruses (en- richment of pathogens out of a mixture of innocuous microbes and human detritus) using a surface-recognition capture, possibly with derivatized magnetic beads;  Spore and cell lysis;  Extraction and purification of nucleic acids;  Possible enrichment and capture of nucleic-acid sequences that are signatures of virulence in human pathogens;  Whatever amplification is necessary (perhaps priming off of the virulence regions);  Fragmentation and size selection, as needed;  Addition of bar codes, as needed;  Ligation, as needed;  Adjustment of buffers, washing, and separation, as needed for every step to produce the necessary sequencing library;  Sequencing with each read being ≈ 400 bases;  Maybe quantification of starting copy number; and  Informatics analysis of sequence, including “identification” of strain and substrain variants (which is particularly tricky for ss- DNA or RNA viruses) resulting in actionable output. INSTRUMENTS, PLATFORMS, AND PROBLEMS There are various issues related to instruments and platforms. Con- cerning polymerase-based assays that use thermal cyclers, for example, if disposable, single-use tubes or wells are used, what burden of consuma- bles is incurred? If flow-through instruments are used, what difficulties will arise in cleaning out all prior material? There are also issues related to the lifetime and aging of the flow-through, thermal-cycling chambers and tubing, such as what happens with repeated exposure to bleach. If fixed surface is used, such as a microarray, how does one integrate the flow system and hybridization chamber—swish the sample back and forth or use diffusion, only? What about the time for equilibration/signal versus the starting copy number and the concentration of possibly inter-

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APPENDIX G 167 fering sequences? There are also questions about the size and cost of each array. What sample preparation is needed, including either nonspecific or specific (i.e., a virulence region) and multiplexed amplification (similar to AmpliSeq®, but targeted to “regions of interest”). Concerning the leak-free insertion of a new array for the next sam- ple, what complexity and reliability of robotics would be required? How about a large microarray with many subarrays in one large fluidic mani- fold, where each microarray is operated individually? What pattern-recognition software exists or needs to be created? What will be need to distinguish multiple targets simultaneously? What about reproducibility and quantification? Would the DHS application require a custom sequencing platform? Existing systems that slowly provide 108 or 109 reads do not seem to be a good match to the DHS workspace. Also, the carryover of reads from prior sequencing runs could prevent simple adoption of an existing se- quencing platform. What choices are there for robotic or microfluidic platforms to per- form sample handling and sample preparation for a customized sequenc- ing instrument? One is IntegenX, but there are others. What software is needed to reduce or eliminate the local need for experienced informatics personnel to interpret sequence data, including such things as natural sequence variability and also instrumental mis- calls? If resident, expert software is feasible, what is its time frame to reach TRL 6? It is this author’s understanding that multiple groups are already working on such software, so a system with custom sequencing and resident, expert software could be available in the 2016–2020 time frame. Performance of both microarray platforms and sequencing platforms would likely benefit significantly from targeted multiplex polymerase amplification of regions of interest. There would be less confounding data and chemistry in the readout step, and the pattern-recognition or base-calling software would have an easier job. FIELD-TESTED AUTONOMOUS SYSTEMS There are a number of mature candidates for field-tested autonomous systems. At TRL 9 is the Northrop Grumman autonomous pathogen de- tector system, which has performed autonomous operations using multi-

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168 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH plexed PCR assays for the DHS for some period with no false-positives and no false-negatives. The general operation has been described by Regan et al. (2008) (see Figure G-2). At TRL 8 is the Hamilton Sundstrand M-BAND PositiveID, which has successfully detected and identified blind samples provided by the DHS, as described by Sanchez et al. (2011) (see Figure G-3). Both the Northrop Grumman system, which collects 1.7 m3/min, and the Hamilton Sundstrand system, which collects 0.4 m3/min, are well beyond the TRL 6 point for field deployment by 2016, assuming no fun- damental changes in their assays. The Northrop Grumman system ap- pears to have an easier path forward to expansion of the number of assays (the liquid array approach has a built-in capability to handle up to 100 target sequences) and runs internal positive and negative controls for every polymerase-amplification reaction. Because of the demand for inexpensive sequencing of individual human genomes, some relevant science and technology continue to advance—not an integrated, fully automated stand-alone system, but good long-term prospects. One example is IntegenX, an automated sam- ple preparation for sequencing (see Figure G-4). FIGURE G-2 Autonomous pathogen detector system.

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APPENDIX G 169 FIGURE G-3 M-BAND. FIGURE G-4 IntegenX.

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170 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH ACKNOWLEDGMENTS This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. The author gratefully acknowledges discussions with Thomas Bunt, Shea Gardner, Staci Kane, Pejman Naraghi-Arani, David Rakestraw, Marilyn Ramsey, Tom Slezak, Mark Wagner, and Lewis Wogan of Law- rence Livermore National Laboratory; John Dzenitis and Phillip Belgrader of BioRad; M. Allen Northup, formerly of Cepheid and MFSI; David Walt, Tufts University; Stevan Jovanovich, IntegenX; Kimothy Smith and Lyle Probst of PositiveID/MFSI; George Dizikes, Illinois De- partment of Public Health; Brent Chyna, State of Illinois and Astrix Technology Group, Inc.; Bernadette Johnson, Lincoln Labs; Cynthia Bruckner-Lea, Pacific Northwest National Laboratory; Michael Farrell, Centers for Disease Control and Prevention; and Darren Link, Raindance. REFERENCES Drmanac, R., I. Labat, I. Brukner, and R. Crkvenjakov. 1989. Sequencing of megabase plus DNA by hybridization: Theory of the method. Genomics 4:114–128. DuBose, A. J., et al. 2013. Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene. Nucleic Acids Research 41(6):e70. Gevensleben, H., et al. 2013. Noninvasive detection of HER2 amplification with plasma DNA digital PCR. Clinical Cancer Research 19(12):3276–3284. Henrich, T. J., S. Gallien, J. Z. Li, F. Pereyra, and D. R. Kuritzkes. 2012. Low- level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR. Journal of Virological Methods 186:68–72. Inglesby, T. V., et al. 2002. Anthrax as a biological weapon, 2002: Updated recommendations for management. Journal of the American Medical Association 287(17):2236–2252. Erratum in 288(15):1849. Jackson, J., et al. 2012. Francisella tularensis Subspecies holarctica, Tasmania, Australia, 2011. Emerging Infectious Diseases 18:1484–1486. Jensen, J. G. 2007. Effect of Atmospheric Background Aerosols on Biological Agent Detectors. Technical report prepared for Headquarters U.S. Air Force/Deputy Director for Counterproliferation.

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