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Institute of Medicine (US) Forum on Microbial Threats. The Science and Applications of Microbial Genomics: Workshop Summary. Washington (DC): National Academies Press (US); 2013.

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The Science and Applications of Microbial Genomics: Workshop Summary.

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Appendix AContributed Manuscripts

A1. THE MICROBIAL FORENSICS PATHWAY FOR USE OF MASSIVELY PARALLEL SEQUENCING TECHNOLOGIES

Authors

Bruce Budowle,^1,2 Sarah E. Schmedes,^1,2 and Randall S. Murch^1,3.

Affiliations

¹ Institute of Applied Genetics, University of North Texas Health Science Center, Fort Worth, TX.

² Department of Forensic and Investigative Genetics, University of North Texas Health Science Center, Fort Worth, TX.

³ Virginia Tech, National Capital Region, Arlington, VA.

The Challenge

Eliminating the threat of terrorist or criminal attacks with microorganisms or toxin weapons is a continual challenge for biodefense and biosecurity programs. The task is difficult for several reasons: (1) the relative ease of access to a variety of effective source materials (Srivatsan et al., 2008) and options for the delivery of a bioweapon, (2) the minute quantities of materials that can be transferred and yet still be effective, (3) the difficulties in detection and analysis of microbiological evidence, and (4) the lack of well-defined approaches regarding credible inferences that can be made from microbial forensic evidence given extant data. At the onset of an event, it may be difficult to distinguish between a deliberate attack and a naturally occurring outbreak of an infectious disease (Morse and Budowle, 2006; Morse and Khan, 2005). Even if evidence strongly supports the hypothesis of a deliberate attack, it may still be very difficult to attribute the attack with certainty to those responsible (i.e., attribution). Attempts to resolve the crime will require advanced methods for characterizing microbial agents, as well as a combination of traditional investigation and intelligence gathering activities.

The Approach

In response to the need to determine the nature of the threat and the source of the weapon and to identify those who perpetrated the crime, the scientific community rose to the occasion beginning in 1996 and developed the field of microbial forensics. Microbial forensics is the scientific discipline dedicated to analyzing evidence from a bioterrorism act, biocrime, hoax, or inadvertent microorganism/toxin release for attribution purposes (Budowle et al., 2003, 2005a; Köser et al., 2012; Morse and Budowle, 2006). Another goal can be to support analysis of potential bioweapons capabilities for counter-proliferation, treaty verification, and/or interdiction. A forensics investigation initially will attempt to determine the identity of the causal agent and/or source of the bioweapon in much the same manner as in an epidemiological investigation. The epidemiological concerns are identification and characterization of specific disease-causing pathogens or their toxins, their modes of transmission, and any manipulations that may have been performed intentionally to increase their effects against human, animal, or plant targets (Morse and Budowle, 2006; Morse and Khan, 2005). A microbial forensics investigation proceeds further in that evidence is characterized to assist in determining the specific source of the sample, as individualizing as possible, and the methods, means, processes, and locations involved to determine the identity of the perpetrator(s) of the attack or to determine that an act is in preparation. A systems analysis may be able to determine the processes used to generate the weapon or how it was delivered, which also can help inform the investigation and attribution decision. The ultimate goal is attribution—to identify the perpetrator(s) or to reduce the potential perpetrator population to as few individuals as possible so investigative and intelligence methods can be effectively and efficiently applied to “build the case” (Figure A1-1).

FIGURE A1-1

The microbial forensics attribution continuum.

Forensic Targets

Microbial forensic evidence may include the microbe, toxin, nucleic acids, protein signatures, inadvertent microbial contaminants, stabilizers, additives, dispersal devices, and indications of the methods used in a preparation. In addition, traditional types of forensic evidence may be informative and should be part of the toolbox of potential analyses of evidence from an act of bioterrorism or biocrime. Traditional evidence includes fingerprints, body fluids and tissues, hair, fibers, documents, photos, digital evidence, videos, firearms, glass, metals, plastics, paint, powders, explosives, tool marks, and soil. Other types of relevant evidence must be considered to exploit avenues to better achieve attribution, including proteins and chemical signatures. These types of signatures can only be obtained from crimes where the weaponized material or delivery device is found; they have little use in covert attacks where the biological agent is derived from the victims. Many of these methods are based on sound technologies and are complementary. They can be combined to identify signatures of sample growth, processing, and chronometry (Morse and Budowle, 2006). Matching of sample properties can help to establish the relatedness of disparate incidents. Furthermore, mismatches might have exclusionary power or signify a more complex causal relationship between the events under investigation. The results of these analyses can provide information on how, when, and/or where microorganisms were grown and weaponized. While the goal of a microbial forensic analysis is to characterize a sample such that it can be traced to a unique source or at least eliminate other sources, it is unlikely that microbial forensic evidence alone is currently adequate to meet this goal.

Emerging Science and Technology

To enhance attribution capabilities with microbial evidence, considerable attention is being invested in molecular genetics, genomics, and bioinformatics. These fields are essential to microbial species/strain identification, fine genome variation, virulence determination, pathogenicity characterization, possible genetic engineering, and attaining source attribution to the highest degree possible. The various tools that have been, or are being, developed in these areas will help to narrow the potential sources from which the pathogen used in an attack may have originated. Indeed, sequencing of an entire genome has been demonstrated as feasible in epidemiological investigations, such as the recent studies of outbreaks of E. coli O104:H4 in Germany and cholera in Haiti (Brzuszkiewicz et al., 2008; Chin et al., 2011; Grad et al., 2012; Hasan et al., 2012; Hendriksen et al., 2011; Mellmann et al., 2011; Rasko et al., 2011; Rohde et al., 2011). In addition, metagenomics studies may become foundational on describing diversity and endemicity. Endemicity becomes important when the relationship between microbes or their genetic residues in samples collected from a site of interest and microbes in the environmental background need to be defined. While the inferential capacity of microbial forensics genetics has yet to reach its full power, the phenomenal new generations of sequencing technology and the concomitant developments for bioinformatics capabilities to handle and extract the explosion of data offer potentials for enhancing microbial forensic investigations. Indeed, the science and technology supporting microbial forensics are advancing at an inconceivable rate. For example, in 2002 in response to the anthrax letter attack, whole genomes of a few isolates were sequenced using shotgun sequencing by TIGR (Budowle et al., 2005b; NRC, 2009; Ravel et al., 2009; Read et al., 2002, 2003). That seemingly nominal analysis, by today’s capabilities, cost approximately $250,000 for one genome, took several weeks, and was unable to characterize but a few samples. Today, such enterprises are a fraction of the cost (and continue to drop dramatically), are becoming more automatable, and provide gigabases and terabytes of data in a matter of days (Bentley et al., 2008; Holt et al., 2008; Loman et al., 2012; MacLean et al., 2009; Margulies et al., 2005).

Given the enhanced capabilities of nucleic acid sequencing of microbes the microbial forensics community will embrace these molecular tools. Although developments are needed, one can envision identification of microbes at the species, strain, and isolate levels being transformed using next- (or better termed “current-”) generation sequencing (CGS). Fine genome detail could become available for routine microbial forensic use. Because CGS provides whole genome characterization capabilities with high depths of coverage (100s to 1,000s fold and beyond), the technology will serve a critical role for research, such as genetic diversity and endemicity studies via metagenomics, and become a rapid diagnostic tool initially when viable and culturable microbes are available. Indeed, whole genome sequencing will reduce the need for a priori design of assays directed at defined species. The technology should apply at some resolution level to any genome without knowledge of the target. In addition, whole genome sequencing offers the capability to evaluate a sample for indications of genetic engineering.

Current Realities

However, not all microbial forensic evidence will present itself in a manner where copious quantities of target are available. Some samples will be highly degraded and/or contaminated. Thus, there will be challenges to extract the most information possible from limited materials and non-viable organisms. To meet these challenges, improved sample collection and extraction methods will be needed, nucleic acid repair methods will be sought, target amplification strategies such as whole genome amplification and selective target capture will be sought, and sequencing chemistries will be enhanced. Because of the throughput, CGS technologies can analyze multiple samples and not even begin to exploit the full throughput of the systems (Brzuszkiewicz et al., 2011; Cummings et al., 2012; Eisen, 2007; Hasan et al., 2012; Holt et al., 2008; Howden et al., 2011; Loman et al., 2012; MacLean et al., 2009; Relman, 2011; Rohde et al., 2011). However, the technology still is evolving and currently does not offer the sensitivity of detection to analyze low-quantity and low-quality DNA samples without some amplification approach prior to sequencing. Nonetheless, CGS is sufficiently mature to be considered useful for microbial forensic applications. Alternatively, technologies, such as mass spectrometry analyses of nucleic acids and real-time PCR, will continue to be used because they offer rapid detection (at species and strain levels) at substantially lower costs (Jacob et al., 2012; Kenefic et al., 2008; Sampath et al., 2005, 2009; U’ren et al., 2005; Vogler et al., 2008).

There are a number of CGS instruments and different chemistries. They include Miseq^® System and Hiseq™ Sequencing Systems (Illumina, Inc., San Diego, CA), Ion Personal Genome Machine™ (PGM™) Sequencer, Ion Proton™ Sequencer and SOLiD^® Systems (Life Technologies, Foster City, CA), and the 454 Genome Sequencer FLX and GS Junior Systems (Roche Diagnostics Corporation, Indianapolis, IN) (Bentley et al., 2008; Cummings et al., 2010; Loman et al., 2012; Margulies et al., 2005). In addition, single molecule detection platforms, such those from Pacific Bioscience (Chin et al., 2011; Eid et al., 2009) and possibly Oxford Nanopore (Branton et al., 2008) are on the horizon. Each system offers some advantages and limitations for sequencing that will need to be defined with considerations of library preparation, read length, and accuracy. The evaluations should be based on the needs of application-oriented laboratories and not necessarily those of a research laboratory. Initially, microbial forensics instruments will be maintained in controlled laboratory environments.

Library preparation is one of the critical limiting factors for transferring CGS technology from a research environment to that of an operational laboratory. Currently, only a few samples can be prepared at any given time. Thus, while the sequencing throughput of the platforms is high, a sufficient number of samples cannot be readily prepared in an appropriate amount of time to meet the full capacity of the system. Library preparation needs to be simplified. Haloplex (Agilent, Santa Clara, CA) is an example of a library preparation process that potentially can reduce the preparation work required (www.halogenomics.com). This library preparation approach is a single-tube target amplification methodology that enables a large number of library samples to be prepared manually. The general process is: (1) restriction digest and denature the sample; (2) hybridize probes to targeted ends of the digested fragments; (3) circularize and ligate the molecules; and (4) introduce bar codes and amplify the targets by polymerase chain reaction (PCR). Eventually with automation the process might accommodate the number of samples that may be encountered by high-throughput operational laboratories. As many as 96 bar codes are available, which fits well with the 96-well format and reduces the preparation time from 2 weeks or several days to 6 hours. However, currently Haloplex is not available for use with non-human nucleic acids. One constraint is that the Haloplex system employs restriction digestion of the DNA. The restriction enzymes can potentially cleave a target site of interest (either a single nucleotide polymorphism (SNP) site or within a repeat motif) and render the marker untypable. Unfortunately, the enzymes used in Haloplex are proprietary, and one cannot readily scan for the restriction sites that would be incompatible with the designated targets (although palindromes can be sought for potential sites that may be obliterated). Another strategy for simplifying library preparation and decreasing sample input is that of the Nextera XT DNA Sample Preparation Kit (www.illumina.com). Strategies, such as the Haloplex system and the Nextera XT DNA kit, hold promise for simplifying and possibly automating library preparation.

Another factor to consider with CGS technology is sequencing read length and accuracy. Current read lengths for the most widely used CGS instruments typically do not exceed 200 bases, and when they do, the quality of base calling decreases substantially along the length of a read. Longer reads with higher accuracy are necessary. Advances in technology for some platform systems suggest that reads up to 400 bases will be feasible in 2012.

Another consideration of platform selection is for situations where rapid responses are required (such as in military operations, some pandemics, and bioterrorism acts). Initially, platforms will be placed in laboratories with controlled environments. One can envision the technology being taken to the field for immediate response and exigent circumstances. Robustness of the instrumentation, supply lines of reagents, and service support will be part of the decision process for the instrumentation/chemistry of choice. Fortunately, the technology and supporting interpretation tools continue to evolve and likely will become more robust.

Seeking More Power and Depth

For design and selection of systems and diagnostics, different diagnostic-based strategies can be considered. They can be based on the sample type, the sample matrix, the amount of work, or the question that one is attempting to address. The latter may be the best suited for conceiving workflow systems. The different scenarios should be considered where nucleic acid analyses may be applied, because these will help guide the needs for the microbial forensic community. They likely are (1) identification of species/strain (i.e., similar to epidemiological needs), (2) attribution, (3) genetic engineering, (4) sample-to-sample comparisons, and (5) metagenomics for endemicity (or a modified metagenomics for sample characterization) (Figure A1-2).

A flow diagram showing the work and information flow from sample to analysis

FIGURE A1-2

A general overview of the work and information flow from sample to analysis to information developed based on use of second-generation sequencing technology.

Sample identification generally would be direct characterization to identify the agent for immediate determination of potential threat and probable cause to investigate further. The process of attribution would drill down to the finest resolution possible and make comparisons to other reference samples, databases, or repositories to reduce the possible sources from which the sample originated or to a recent common ancestor. Genetic engineering could be detected by whole genome sequencing.

Metagenomics studies have been performed on several platforms, and they will likely provide some foundational data on diversity and endemicity (Eisen, 2007; Relman, 2011; Tringe et al., 2005). The value could be searching various niches for select agents. Suppose that in every sample tested certain select agents are identified. Then there can be two consequences: one is that it may be more difficult to elucidate natural outbreaks versus intentional releases (although strain resolution may reduce the uncertainty); the second could be that such high resolution may be less informative at some threshold depth of coverage.

Most metagenomic work to date has been by exploiting a small, single sequence target (16s rRNA), at a very high depth of coverage (Rusch et al., 2007; Venter et al., 2004). These studies often cannot provide resolution beyond family to genus levels. Clearly such broad range definition will not enable individualization or identify select agents. The anthrax investigation could have benefited from a modified metagenomics characterization. The putative common source of the material (RMR1029) was composed of a population of very similar cells. The colony morphological variants found in the evidence from the 2001 anthrax letter attacks were minority components and because of sample preparation and stochastic effects the minor variants potentially could be difficult to detect with PCR-based assays that were developed for the investigation. Because of the high depth of coverage with CGS, the population of low-level variants may be more readily detected, especially if an amplification enrichment step was included that focused only on the known variant sites that defined the morphology types. Such high depth of coverage would substantially reduce the false-positive rate and improve confidence in the potential relationship of the most similar samples to focus investigative leads (Cummings et al., 2010). Indeed, the depth of coverage could be in the millions. While exquisitely sensitive, platform- and chemistry-specific errors may confound interpretation, and thus thresholds of reliability may be necessarily invoked.

One could envision extending this population depth analysis, which in essence is a simplified metagenomic analysis, and exploiting the concept of using a multi-locus sequence typing (MLST) approach to provide a species-level identification capability (Maiden et al., 1998; Spratt, 1999). A few loci (perhaps the seven typically applied to MLST to 15) could be selected as a standard (e.g., for bacteria). If there is a combination of sufficiently stable sites and evolutionarily rapid sites, the loci could indicate species- to strain-level presence in mixed and metagenomic samples. Using the core seven used for MLST could allow some questions regarding time and place of isolation, host or niche, serotype, and some clinical or drug resistance profiles. This will not be a trivial process because each of the sites will not be physically linked. However, one could determine, if the complete set or a reasonable subset of targets are in a sample, whether there is confidence that a particular species or sets of species are present. In theory this approach could be extended to strain levels. There certainly is enough throughput to consider this capability. The potential already has been established with electrospray ionization mass spectrometry of targeted genes for rapid bacterial species identification (and even for viruses such as influenza). There are sufficient bacterial genomes that have been sequenced to test our hypothesis, and work is under way.

Inferences about the significance of genetic evidence may not reach the ultimate goal of attribution. The most confounding constraint on reaching the full power of attribution is scant data on diversity and endemicity. The vast diversity of the microbial world is unknown and will not be defined substantially with current approaches in the area where a biocrime or bioterrorist attack has occurred. This limitation is not the sole purview of the microbial forensics community; it plagues the epidemiologists as well. Another limitation that evidentiary samples will likely have is an unknown history. Lack of knowledge on how it was manipulated (e.g., number of passages, exposure to mutagenic agents, length of storage) will complicate providing inferences about the significance or strength of sequencing results, especially because the distance between samples will be determined by the degree of similarity or dissimilarity. Indeed, even defining what is a “match” or “similar” may not be straightforward. Keim (personal communication) has stressed this uncertainty and proffered new terminology—a “member,” to the microbial forensic lexicon based on phylogenetics for the relationship of a sample to some reference samples. Regardless of the terminology used, some data will be needed to define the uncertainty of a “membership” or “association.” In 2006, the need for reconciliation between microbial genomics and systematics was described; microbial forensics and epidemiology were seen to offer useful, practical venues to frame the gaps and priorities (Buckley and Roberts, 2006). This challenge remains.

Some assessment of the strength or significance of an analytical result and subsequent comparison also is needed (Budowle et al., 2008; Chakraborty and Budowle, 2011). Of course, because of scant supporting data, such an endeavor will be challenging. Qualitative and/or quantitative statements of the significance of the finding will need to be developed. As an example, consider a forensic analysis of whole genome sequence data that compared two or more sequences, such as an evidence sample profile with that of a reference sample that may be considered a possible direct link or have a common ancestor. The evolutionary rates of the variants will need to be known. But perhaps as consequential, sequencing error and other factors could inflate the dissimilarity between samples and add a degree of “uncertainty” to some extent. Thus, efforts in defining and quantifying the error rates associated with each CGS platform and chemistry are critically important.

Beyond comparison of samples for identification purposes are inferences by whole genome sequencing of phenotypic (i.e., functional) properties of a microbe. For example, even with a whole genome sequence whether a microbe phenotypically displays antimicrobial resistance or susceptibility is still limited. Bacteria may contain multiple pathways, and how the different genes interact is far from being completely understood (Eisen, 2007; Köser et al., 2012; Relman, 2011). Substantial research will be needed such that genotype can be used reliably to predict phenotype.

Making Sense of Data

The ever-increasing amount of microbial genomic sequence data presents a variety of challenges related to the handling and storage of data and the development of bioinformatics methods that can accommodate such large numbers of whole genomes. Being able to analyze the vast amounts of data in a timely fashion is a key challenge to leveraging the power of these newer sequencing platforms. Software, hardware, and IT support may be the greatest barrier to use of CGS technology. It is unlikely that dedicated bioinformaticists will reside in every microbial forensics laboratory. Data cannot be sent to web-based clouds and be analyzed because the results may be classified. Instead, some standardization and standard operating data analysis and interpretation approaches will be needed. Pipeline and interpretation software will need to be evaluated for reliability and seamless diagnostic flow without bioinformatics expert intervention. The output of results must be intelligible to the microbial forensic analyst as well. The ideal software should be a comprehensive tool(s) enabling microbe detection to determination of engineering.

The government should rely heavily on industry and well-established genome centers. The commercial competitive environment is driving down costs and improving informatics pipelines without the need for extensive investment. Leveraging these efforts will help meet the needs of microbial forensics more expeditiously than going it alone. The centers (to include the national laboratories) are evaluating platforms and chemistries and are generating data at unprecedented levels. They are providing solutions to massive data handling, including storage, curation of reference data, annotation, and data analysis.

Collection and databases are needed to house the microbial genomic data and when possible the accompanying meta-data. No standards yet exist for building databases to meet the needs of the microbial forensic community. Requirements for storage and retrieval of raw sequence data in microbial forensics cases and supporting inferential data must be developed. Given the high throughput and anticipated speed of analyses, it is conceivable that meaningful databases can be developed “on the fly” that better reflect the diversity where the crime was committed (to include the preparation laboratory to the crime scene).

The power of microbial forensics techniques, tools, software, and databases that are used need to be understood, and their limitations even more so need to be understood. To achieve this goal methods need to be validated, and validation should be a requisite of any forensic repertoire. Indeed the forensic sciences in general are facing well-deserved criticism for not necessarily having sound foundations and overstating the strength of the evidence (NRC, 2009). Attempts to attribute any attack to a person(s) or group should rely on acurate and credible results. The interpretation of such results might seriously impact the course or focus of an investigation, thus affecting the liberties of individuals or even being used as a justification for a government’s military response to an attack or threat of an attack. Therefore, the methods for collection, extraction, and analysis of microbial evidence that could generate key results need to be as scientifically robust as possible, so the methods can be high performing and the results defensible for decision makers and to the legal, international government, law enforcement, and scientific communities, as well as scrutiny by the media.

Validation Is Essential

Validation is frequently used to connote confidence in a test or process, but it may be better thought of as defining the limitation of a method, process, or assay (Budowle et al., 2003, 2006, 2008). It still is common for the term validation to be used vaguely or to remain undefined when applied to process performance evaluation. The degree of validation varies from nominal to rigorous. The consequences of such varied requirements can be catastrophic if methods used in microbial forensic investigations are poorly constructed, under-developed, or generate results that are difficult to interpret. The validation process needs to be defined as to what is expected to be achieved by a validation study.

Validation determines the limits of a test. It does not mean that a test must be 100 percent accurate or have no cross-reactivity, false-positive results, or false-negative results to be considered useful. It is often thought of as a process applied to the analytical portion of a system. This concept is only partly correct. The limits that the methods can provide must be demonstrated and documented for all steps of the process to include sample collection, preservation, extraction, analytical characterization, and data interpretation. Furthermore, it is recognized that as new technologies and capabilities are developed to address the needs of the microbial forensics community, key principles and performance parameters including accuracy, precision, bias, reliability, sensitivity, and robustness will need to be determined. Robust quality assurance and data control systems are required to achieve confidence in results by diverse users of the information. It is imperative that both technical and interpretation limitations (and thus accuracy and error) be defined. Additionally, a key resource for microbial forensic research, validation, and analysis is access to well-defined and curated microbial collections and data sets that are as comprehensive as is possible to the task. This effort includes the structure, content, and quality of the data sets. While some collections have been started for use in research, or created for case-specific use, no comprehensive repository exists to support microbial forensics, and standards are not codified for meta-data and data curation.

The implications of highly technical data, epidemiological data, traditional evidence data, and investigative or intelligence information are complex and need to be appreciated for their strengths and limitations. Because scientific data can affect the decision-making process for retaliation, preemptive actions, and/or courtroom deliberations, it is imperative that those directly involved in microbial forensics or those who may use the results for investigative lead value or more direct associations be properly educated (or at least properly apprised) of the implications of such data. To meet this necessary goal, education and training are critical to disseminate the principles, development, and applications of the evolving field of microbial forensics. Educational strategies and programs need to be constructed and training programs developed on the varied scientific foundations that support microbial forensics.

If validation processes are not defined and not followed and proper training or communication is not provided, then it is possible that a false sense of confidence may be associated with a poor method or process or from a result of limited significance. There are myriad methods, processes, targets, platforms, and applications. Yet some basic requirements transcend individual differences in methods, and these can be reinforced by contextual description (Table A1-1). Validation needs to be codified. Efforts are under way and should be applied equally across the user space.

TABLE A1-1

Validation Criteria List.

Conclusion

Microbial forensics should embrace and validate newly developed and emerging molecular biology technologies and phylogenetics approaches, and pursue potential forensic information and comparative sources, such as might be achieved through metagenomics. Genetic analyses of microorganisms often are a powerful tool for differentiating species, isolates, and strains. Similar to human DNA forensic identification, DNA sequences of microorganisms can be used to identify and differentiate between isolates and strains of a single microbial species; however, nucleic acid–based identification is not as resolving with respect to source attribution in microbial forensics as with human DNA forensic analysis. The basic constituents of nucleic acids essentially are the same for bacteria and humans; however, unlike humans, bacteria, viruses, and fungi multiply rapidly in a clonal fashion and can readily share or exchange genetic material between and among species. These differences and uncertainties due to scant supporting data must be taken into consideration during analysis, interpretation, and reporting related to the findings derived from microbial genetic evidence. For the foreseeable future the ability of microbial forensics to establish that a sample collected from either a crime scene or a person of interest can be attributed to a known source to a high degree of scientific certainty will be limited. Therefore, the methods must be reliable and robust, and the uncertainty associated with any interpretation should be properly conveyed.

Microbial forensics experts and those who contribute in closely related fields need to work together to advance the science, to validate methods to scientific and legal standards, and to transition interpretation of results and conclusions from such analyses into something that can be used by the criminal justice system, the policy community, and other stakeholders. It is incumbent upon the microbial forensics community to make every effort to interpret and communicate objectively and effectively the advantages and limitations of both microbial forensics and traditional forensic science analyses. Consumers of microbial forensic information who incorporate this evidence into decision making should be provided accurate, reliable, credible, and defensible results, interpretations, and context.

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