Forensic Attribution Research Articles

Integrated epigenomic exposure signature discovery.

Aim: The epigenome influences gene regulation and phenotypes in response to exposures. Epigenome assessment can determine exposure history aiding in diagnosis.Materials & methods: Here we developed and implemented a machine learning algorithm, the exposure signature discovery algorithm (ESDA), to identify the most important features present in multiple epigenomic and transcriptomic datasets to produce an integrated exposure signature (ES).Results: Signatures were developed for seven exposures including Staphylococcus aureus, human immunodeficiency virus, SARS-CoV-2, influenza A (H3N2) virus and Bacillus anthracis vaccinations. ESs differed in the assays and features selected and predictive value.Conclusion: Integrated ESs can potentially be utilized for diagnosis or forensic attribution. The ESDA identifies the most distinguishing features enabling diagnostic panel development for future precision health deployment.

Advancement in oil forensics through the addition of polycyclic aromatic sulfur heterocycles as biomarkers in diagnostic ratios

In current oil spill forensics, diagnostic ratios of hydrocarbon biomarker responses are commonly used to compare oil spill samples to source materials in order to determine the identity of the oil. This well recognized procedure was developed by the European Committee for Standardization (CEN) with corresponding published EN 15522–2 Oil Spill Identification guidelines. However, it is further recognized that weathering can have a negative effect on some of the biomarkers used in the analysis, leading to decreased confidence in the result. In this study, polycyclic aromatic sulfur heterocycles (PASHs) and their alkylated forms (APASHs) were assessed for their potential as additional biomarkers. With the aim of identifying stable PASHs and APASHs useful as weathered oil biomarkers, the superior specificity of gas chromatography with high resolution mass spectrometry was exploited to determine chromatographic peak responses for sixteen petroleum oil samples. Extensive study, involving microcosm extreme weathering and spreadsheet development, led to the identification of 19 new diagnostic ratios based on newly discovered stable PASH and APASH biomarkers. Application of the extended diagnostic ratio suite showed high potential to improve the forensic attribution of post-spill weathered oil back to its original source.

Speech Portrait of Personality as a Tool of Forensic AuthorshipAttribution

The article discussesapplication of speech portraitpotential in forensic authorship attribution. An illustrative example is the speech portraits of Russian vernacular native speakers of different ages and genders living in the same language community - the village of Kamyshenka, Altai Krai. The results of the study can be used for speech identification within the framework forensic authorship attribution, since the described lexical and grammatical features of the speech of each respondent form unique combinations that allow for the identification procedure. The nature of the narrative strategies in the structure of the speech portrait is also established, which are no less, if sometimes even a more informative feature of the speech portrait of a linguistic persona than the lexical and grammatical features of the speech.

Classification of fentanyl precursors by multivariate analysis of low-field nuclear magnetic resonance spectroscopy data

High-field Nuclear Magnetic Resonance (NMR) spectroscopy combined with multivariate analysis techniques have been used extensively in several fields particularly, metabolomics and food quality assessment. In contrast, low-field Fourier transform NMR instruments are a relatively new analytical platform and recent studies have shown its potential application for forensic narcotic identification. In this study Principal Component Analysis (PCA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) were applied on spectra data from both low-field (43 MHz) and high-field (300 MHz) NMR of two key precursors to fentanyl, N-phenethyl-4-piperidone (NPP) and 4-anilino-N-phenethylpiperidine (ANPP). Three different methods were employed for the synthesis of these two precursors – the Siegfried, Valdez and One-Pot methods. The application of low-field NMR spectroscopy combined with multivariate analysis allowed successful classification of fentanyl precursors, NPP and ANPP, based on each synthetic method’s unique impurity profile. This approach supports the possibility of low-field NMR being employed by law enforcement for forensic attribution of clandestine fentanyl and its precursors to a specific synthetic method.

Negative result: Chemical forensic attribution of ricin preparations using fatty acids analysed with gas chromatography-mass spectrometry

Here, samples of castor oil were extracted from Australian Ricinus communis seeds. The extracted oils were esterified and the derivatives characterised by gas chromatography-mass spectrometry (GC–MS). The fatty acid composition of the oils was profiled, enabling the trace fatty acid components of castor oil to be reported for the first time. The resulting data were also subjected to statistical analysis to test the applicability for provenance and geographic attribution purposes, but no discrimination was found.

Highly accurate classification of biological spores by culture medium for forensic attribution using multiple chemical signature types and machine learning.

Future proliferation of biological expertise and new technology may increasingly lower the difficulty to produce biological organisms for misuse. Rapid attribution of a biological attack is needed to quickly identify the person or lab responsible and prevent additional attacks by enabling the apprehension of suspects. Here, triplicate batches of Bacillus anthracis Sterne strain (BaSt) spores were grown in a total of seven amateur and professional media. Multiple orthogonal analytical signatures (peptides, metabolites, lipids by fatty acid methyl ester (FAME) analysis, bulk organic profile, and trace elements) were collected from the BaSt spores. The proteomics and metabolomics analyses identified promising attribution signature compounds that are unique to each of the seven production methods. In addition, while each of the signature types showed varying degrees of value individually for attributing BaSt spores to the culture medium used to prepare them, fusing results from all five signatures types to increase sourcing robustness and using a random forest sourcing algorithm yielded 100% hold-one-batch-out cross-validation classification accuracy and an average relative source probability for the correct source 5.5× higher than the most probable incorrect source. These preliminary results provide a proof-of-concept for the development of forensic examinations that can attribute biological agents to production methods for use in future investigations.

Part 2: Forensic attribution profiling of Russian VX in food using liquid chromatography-mass spectrometry

This work is part two of a three-part series in this issue of a Sweden-United States collaborative effort towards the understanding of the chemical attribution signatures of Russian VX (VR) in synthesized samples and complex food matrices. In this study, we describe the sourcing of VR present in food based on chemical analysis of attribution signatures by liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with multivariate data analysis. Analytical data was acquired from seven different foods spiked with VR batches that were synthesized via six different routes in two separate laboratories. The synthesis products were spiked at a lethal dose into seven food matrices: water, orange juice, apple purée, baby food, pea purée, liquid eggs and hot dog. After acetonitrile sample extraction, the samples were analyzed by LC-MS/MS operated in MRM mode. A multivariate statistical calibration model was built on the chemical attribution profiles from 118 VR spiked food samples. Using the model, an external test-set of the six synthesis routes employed for VR production was correctly identified with no observable major impact of the food matrices to the classification. The overall performance of the statistical models was found to be exceptional (94%) for the test set samples retrospectively classified to their synthesis routes.

Potential usefulness of SNP in the 16S rRNA gene serving as informative microbial marker for forensic attribution

The field of microbial forensics focuses on the attribution of numerous microbes used by perpetrators inbiocrime and bioattack, including many foodborne pathogens such as Escherichia coli O157:H7 caused serious zoonotic disease. Recent source tracing analysis with the 16S rRNA gene, even genome-wide variations of a pathogen, is increasing as sequencing technologies advance. By using whole genome sequence analysis, more SNPs have been used to create a very finely resolved phylogenetic tree for discriminating close evolutionary relationship among strains. In our study, 1401bp of 16S rRNA gene in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains were sequenced. The result showed that nucleotide transition and nucleotide transversion happened at 14 positions of sequence in the research strains, and variation of seven positions was located at one of nine separate hypervariable regions (V7). Another SNP within the constant region of 16S rRNA gene was also observed as the previous studies. Through comparing the available sequences obtained from the NCBI database, SNP was the only 16S rRNA polymorphism detected among the closely related EHEC O157:H7 stains. Our investigation demonstrates that unique SNPs in 16S rRNA gene have potential discriminatory power as other species-/strain-specific informative markers for attribution purpose.

Targeted Next-Generation Sequencing for Diagnostics and Forensics.

Pathogen-specific, targeted assays such as real-time PCR are generally used for clinical diagnosis of infectious and biothreat diseases as highlighted in the recent West African Ebola virus outbreak where real-time PCR was the primary diagnostic for critical treatment decisions. However, current diagnostic algorithms rarely require determination of a single etiologic agent outside the context of an outbreak response but rather involve interrogation across a spectrum of pathogens with similar pathology and/or epidemiology. The necessity for multiple pathogen interrogation exposes the critical gap in availability of higher-multiplex diagnostics. This caveat is exacerbated with biothreat agents where initial symptoms of an infection are often similar to a myriad of less consequential pathogens. Development of diagnostic platforms is a daunting challenge given the plethora of human pathogens spanning both infectious and biothreat diseases. In general, infectious disease diagnostics must meet defined metrics for performance in clinical matrices as well as show validated efficacy for use in an actionable context to impact patient care. Detection of virulence markers to discriminate microbiota from pathogenic events and the ability to detect the spectrum of pathogen heterogeneity are critical aspects to an ideal diagnostic platform. Biothreat diagnostics include all of these requirements in addition to the need for microbial forensics/attribution. The detection of a biothreat incident (e.g., Bacillus anthracis ), environmental or clinical, has substantial consequences including public angst as well as mobilization of military, public health, and law enforcement responses, the latter of these responses requiring forensic attribution. Detection of both clinical and environmental biothreat incidents remains a perennial issue that requires some level of preparedness to mitigate situations proactively. Next-generation sequencing (NGS)2 offers a potential solution to challenges for both diagnostic and environmental detection of infectious disease and biothreats. Specifically, NGS has the potential to sequence all nucleic acids within a sample, allowing for near …

Taxonomic characterization and forensic attribution of bacillus threat agents using extracellular lipid profiles

Taxonomic characterization and forensic attribution of bacillus threat agents using extracellular lipid profiles

Chemical analysis of bleach and hydroxide-based solutions after decontamination of the chemical warfare agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX)

Detailed chemical analysis of solutions used to decontaminate chemical warfare agents can be used to support verification and forensic attribution. Decontamination solutions are amongst the most difficult matrices for chemical analysis because of their corrosive and potentially emulsion-based nature. Consequently, there are relatively few publications that report their detailed chemical analysis. This paper describes the application of modern analytical techniques to the analysis of decontamination solutions following decontamination of the chemical warfare agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX). We confirm the formation of N,N-diisopropylformamide and N,N-diisopropylamine following decontamination of VX with hypochlorite-based solution, whereas they were not detected in extracts of hydroxide-based decontamination solutions by nuclear magnetic resonance (NMR) spectroscopy or gas chromatography-mass spectrometry. We report the electron ionisation and chemical ionisation mass spectroscopic details, retention indices, and NMR spectra of N,N-diisopropylformamide and N,N-diisopropylamine, as well as analytical methods suitable for their analysis and identification in solvent extracts and decontamination residues.

Preliminary Evaluation of the Field and Laboratory Emission Cell (FLEC) for Sampling Attribution Signatures from Building Materials

Clandestine laboratories [e.g., drug, explosive, or chemical warfare (CW)] can be processed for chemical evidence that identifies the synthetic targets and reaction paths. Further, trace impurities contained in the product constitute a fingerprint that can correlate laboratories, associate distributed product (i.e., a terrorist CW attack) with the production laboratory, or relate various attacks. This phenomenological study evaluates the field and laboratory emission cell (FLEC) for its suitability for sampling volatile attribution signatures. Dissipation of a wide range of attribution signature representatives was studied from a variety of typical building materials using FLEC sampling. Results showed rapid and near complete analyte evaporation from metal, intermediate permanence on Teflon, and strong retention on vinyl tiles. FLEC also proved useful for sampling residues left after dissipation of a sulfur mustard stimulant. In summary, preliminary evaluation of FLEC for forensic attribution applications looks promising. Advantages, practical considerations, and disadvantages of FLEC sampling are discussed.

The use of textual, grammatical and sociolinguistic evidence in forensic text comparison:

This article suggests the usefulness of the concept of ‘idiolectal style’ in forensic text comparison and presents evidence that was used by the defence in a forensic authorship attribution case to show that the author of four non-disputed faxes could also be the author of four disputed emails, both sets written in Spanish. Apart from qualitative textual analysis, two different quantitative approaches were undertaken: a) the use of the concepts of markedness and saliency in order to establish the rarity in the frequency and use of two grammatical variables - by comparing their behaviour to the one observed in a general corpus - indicated in both data sets the favourite use of two variants - redundant yo and compound periphrastic relative pronoun el cual, and b) the statistical analysis of sequences of linguistic categories classified and grouped the texts in the two sets under analysis (and also a control text set from another real forensic case) very closely, showing their discriminatory potential, in particular when the sequences were bigrams, something which would lead to the conclusion that these two text sets were produced by the same author.

Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing

BackgroundIn the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization.ResultsTo demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare false-positive SNPs were associated with variable nucleotide tandem repeats.ConclusionsThe high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution.

Use of Parent Polycyclic Aromatic Hydrocarbon (PAH) Proportions to Attribute PAH Sources in Sediments: A Case Study from the Pacific Northwest

Since the late 1970s, a large body of scientific literature has developed focused on attributing sources of polycyclic aromatic hydrocarbons (PAHs) in sediments. Forensic evaluation of sources of PAHs in sediments has typically relied on the use of the ‘expanded PAH’ analysis in which ‘parent’ (non-alkylated) and alkylated members of each homologous series are quantified and reported. Use of this method for source attribution is widely documented in the peer-reviewed literature and has gained wide acceptance in the forensics community. Although the expanded PAH analysis is the preferred method of using PAH data for source attribution, these detailed forensic data are often not collected during routine environmental site investigations. Conversely, the 16 United States Environmental Protection Agency “priority pollutant” PAHs, which include the parent PAH of each homologous series, are routinely analyzed for and reported. Because of the large number and relatively high costs of sediment and soil remediation projects focused on PAHs, there is significant interest in the use of these parent PAH data for forensic source attribution. The peer-reviewed literature contains a few case studies where parent PAHs have been used to attribute sources, including distinguishing between petrogenic (petroleum) and pyrogenic (combustion and pyrolytic) sources, and among multiple pyrogenic sources. This article focuses on a case study that demonstrates the use of parent PAH ratios or proportions for source attribution for PAHs in sediments offshore of a former wood treating facility located in a highly industrialized port of the northwestern United States. The wood-treating facility was assumed as the source for all of the PAHs present in the sediments; this assumption was questioned in the litigation brought to allocate costs for the sediment remediation. Because the sediment remediation was complete, additional sampling for sophisticated forensic testing was not possible; therefore, the forensics evaluation had to rely on the pre-existing parent PAH data. Using the forensic parent PAH proportions and ratios developed by Yunker et al. (2002) and those of Costa and Sauer (2005), PAHs in the sediments were categorized into those attributed primarily to the wood treating facility creosote and those attributed primarily to a mixture of pyrogenic and petrogenic sources common in the “urban background” of industrialized waterways. This approach provided a basis to propose cost allocation for each of the sediment areas that required remediation.

NOSQL Databases: Forensic Attribution Implications

NoSQL databases have gained a lot of popularity over the last few years. They are now used in many new system implementations that work with vast amounts of data. Such data will typically also include sensitive information that needs to be secured. NoSQL databases are also underlying a number of cloud implementations which are increasingly being used to store sensitive information by various organisations. This has made NoSQL databases a new target for hackers and other state sponsored actors. Forensic examinations of compromised systems will need to be conducted to determine what exactly transpired and who was responsible. This paper examines specifically if NoSQL databases have security features that leave relevant traces so that accurate forensic attribution can be conducted. The seeming lack of default security measures such as access control and logging has prompted this examination. A survey into the top ranked NoSQL databases was conducted to establish what authentication and authorisation features are available. Additionally the provided logging mechanisms were also examined since access control without any auditing would not aid forensic attribution tremendously. Some of the surveyed NoSQL databases do not provide adequate access control mechanisms and logging features that leave relevant traces to allow forensic attribution to be done using those. The other surveyed NoSQL databases did provide adequate mechanisms and logging traces for forensic attribution, but they are not enabled or configured by default. This means that in many cases they might not be available, leading to insufficient information to perform accurate forensic attribution even on those databases.