An in-field evaluation of rapid DNA instruments for disaster victim identification.

In 2019 and 2020, disaster victim identification (DVI) simulations were conducted at the Australian Facility for Taphonomic Experimental Research. Whole and fragmented cadavers were positioned to replicate a building collapse scenario and left to decompose for up to 4 weeks. This study evaluated the utility of the ANDE™ 6C Rapid DNA System and the RapidHITTM ID System for DVI in the field and mortuary. Applying post-mortem nail and tissue biopsy samples showed promise, with the added benefit of minimally invasive collection procedures and limited preparation requirements. The preferred platform will depend on a number of factors, including its intended use and operating environment.

The QIAGEN 140-locus single-nucleotide polymorphism (SNP) panel for forensic identification using massively parallel sequencing (MPS): an evaluation and a direct-to-PCR trial.

Massively parallel sequencing (MPS) of identity informative single-nucleotide polymorphisms (IISNPs) enables hundreds of forensically relevant markers to be analysed simultaneously. Generating DNA sequence data enables more detailed analysis including identification of sequence variations between individuals. The GeneRead DNAseq 140 IISNP MPS panel (QIAGEN) has been evaluated on both the MiSeq (Illumina) and Ion PGM™ (Applied Biosystems) MPS platforms using the GeneRead DNAseq Targeted Panels V2 library preparation workflow (QIAGEN). The aims of this study were to (1) determine if the GeneRead DNAseq panel is effective for identity testing by assessing deviation from Hardy-Weinberg (HWE) and pairwise linkage equilibrium (LE); (2) sequence samples with the GeneRead DNAseq panel on the Ion PGM™ using the QIAGEN workflow and assess specificity, sensitivity and accuracy; (3) assess the efficacy of adding biological samples directly to the GeneRead DNAseq PCR, without prior DNA extraction; and (4) assess the effect of varying coverage and allele frequency thresholds on genotype concordance. Analyses of the 140 SNPs for HWE and LE using Fisher's exact tests and the sequential Bonferroni correction revealed that one SNP was out of HWE in the Japanese population and five SNP combinations were commonly out of LE in 13 of 14 populations. The panel was sensitive down to 0.3125 ng of DNA input. A direct-to-PCR approach (without DNA extraction) produced highly concordant genotypes. The setting of appropriate allele frequency thresholds is more effective for reducing erroneous genotypes than coverage thresholds.

Bacillus species at the Canberra Airport: A comparison of real-time polymerase chain reaction and massively parallel sequencing for identification.

Anthrax, caused by the Gram-positive, spore forming bacterium Bacillus anthracis, is a disease with naturally occurring outbreaks in many parts of the world, primarily in domestic and wild herbivores. Due to the movement of people and stock, B. anthracis could, however, be at transportation hubs including airports. The continuous threat to national and international security from a biological agent release, or hoax attack, is a very real concern. Sensitive, robust and rapid (hours-day) methods to identify biological agents, including B. anthracis, and distinguish pathogenic from non-pathogenic species, is an essential cornerstone to national security. The aim of this project was to determine the presence of Bacillus species at the Canberra Airport using two massively parallel sequencing (MPS) approaches and compare with previous results using real-time polymerase chain reaction (qPCR). Samples were collected daily for seven days each month from August 2011-July 2012 targeting movement of people, luggage and freight into and out of the Canberra Airport. Extracted DNA was analysed using qPCR specific for B. anthracis. A subset of samples was analysed using two MPS approaches. Approach one, using the Ion PGM™ (Thermo Fisher Scientific; TFS) and an in-house assay, targeted the two B. anthracis virulence plasmids (cya and capB genes) and a single conserved region of the 16S rRNA gene. Approach two, using the Ion S5™ (TFS) and the commercial Ion 16S™ Metagenomics Kit (TFS), targeted multiple regions within the bacterial 16S rRNA gene. Overall there was consistency between the two MPS approaches and between MPS and qPCR, however, MPS was more sensitive, particularly for plasmid detection. Whilst the broad-range 16S genomic target(s) used in both MPS approaches in this study was able to generate a metagenomic fingerprint of the bacterial community at the Canberra Airport, it could not resolve Bacillus species beyond the level of the Bacillus cereus group. The inclusion of B. anthracis virulence plasmid targets in the in-house assay did allow for the potential presumptive identifications of pathogenic species. No plasmid targets were in the Ion 16S™ Metagenomics Kit. This study shows the choice of target(s) is key in MPS assay development and should be carefully considered to ensure the assay is fit for purpose, whether as an initial screening (presumptive) or a more specific (but not entirely confirmatory) test. Identification approaches may also benefit from a combination of MPS and qPCR as each has benefits and limitations.

MAPlex - A massively parallel sequencing ancestry analysis multiplex for Asia-Pacific populations.

Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.

Current and emerging tools for the recovery of genetic information from post mortem samples: New directions for disaster victim identification.

DNA profiling has emerged as the gold standard for the identification of victims in mass disaster events providing an ability to identify victims, reassociate remains and provide investigative leads at a relatively low cost, and with a high degree of discrimination. For the majority of samples, DNA-based identification can be achieved in a fast, streamlined and high-throughput manner. However, a large number of remains will be extremely compromised, characteristic of mass disasters. Advances in technology and in the field of forensic biology have increased the options for the collection, sampling, preservation and processing of samples for DNA profiling. Furthermore, recent developments now allow a vast array of new genetic markers and genotyping techniques to extract as much genetic information from a sample as possible, ensuring that identification is not only accurate but also possible where material is degraded, or limited. Where historically DNA profiling has involved comparison with ante mortem samples or relatives, now DNA profiling can direct investigators towards putative victims or relatives, for comparison through the determination of externally visible characteristics, or biogeographical ancestry. This paper reviews the current and emerging tools available for maximising the recovery of genetic information from post mortem samples in a disaster victim identification context.

A SNaPshot of next generation sequencing for forensic SNP analysis.

Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot(®), a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30-40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies' Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7%) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.

Alternative analysis of BOD removal in subsurface flow constructed wetlands employing Monod kinetics.

A new, mechanistic approach for design and analysis of subsurface flow (SSF) constructed wetlands is presented. The model is based on the assumption that the biological processes in wetlands, like other biological systems, exhibit Monod kinetics. A Monod approach fits well with observed wetland performance. It predicts first-order behaviour at low concentrations, that is, pollutant removal rates which increase with increasing pollutant concentration; and zero-order or saturated behaviour at high pollutant concentrations, that is, a maximum pollutant removal rate. A kinetic analysis of subsurface flow constructed wetlands exhibiting Monod kinetics reveals that loading rate, as well as the zero-order degradation rate constant, are essential parameters for efficient wetlands design for the removal of organic carbon. In particular, Monod kinetics enables the identification of an absolute maximum removal rate which is necessary to prevent undersizing in design. This is significant because it represents a theoretical upper bound on loading rate for wetlands design. The analysis is applied to wetlands data collected in North America by the US EPA in order to extract design criteria for BOD removal. It reveals that maximum loadings for SSF wetlands are at least 80 kg ha-1 d-1 for BOD. In addition, a new dimensionless performance efficiency parameter, omega, is presented as a more effective means of comparing wetland performance.

Inter-relationship between adsorption and pH in peat biofilters in the context of a cation-exchange mechanism.

A mathematical model of biofiltration McNevin and Barford (1998) has been augmented to include speciation, acid/base equilibria and pH dependence of adsorptive equilibria. It accurately predicts qualitative aspects of dynamic transients observed in an experimental perfusion column and supported a mechanism of adsorption by cation exchange with acidic functional groups on the surface of peat. It mirrored the buffering capacity of peat when solutions of high and low pH flow over the peat surface. This is a direct result of cation exchange where adsorption of cations increases with pH. This buffering capacity makes peat an attractive medium for engineered biological systems which must often operate within narrow pH bands to optimise biological activity.

Indicators of biofilm development and activity in constructed wetlands microcosms.

Methods to measure protein, exopolysaccharide, viable cell number and INT reduction activity were tested on biofilm growing in a wastewater batch reactor. They were shown to be meaningful indicators of biofilm growth and correlated well with each other. Protein, exopolysaccharide, viable cells and INT reduction rates increased linearly over time. Viable cell number exhibited strong linear correlations with protein (R2= 0.98) and exopolysaccharide (R2= 0.99) while INT reduction rate was somewhat less well correlated (R2= 0.90). Our results indicate production rates of 0.91 x 10(-7) microg EPS per viable cell and 1.0 x 10(-7) microg protein per viable cell. Protein and polysaccharide specific INT reduction rates decreased by approximately 50%, whereas viable cell specific INT reduction rates decreased by 65% and the protein to polysaccharide ratio stayed relatively constant at between 1.1 and 1.2 as the biofilm developed. Measurement of protein, polysaccharide, viable cells and INT reduction rate at depth within the bioreactor showed that they were concentrated in the top 1cm of the influent end of the reactor and each decreased to a base level within 4.5 cm of the inlet. Protein to polysaccharide ratios increased with depth in the reactor and the specific INT reduction rates were maximal at 4.5 cm depth. The results indicate that the biomass can take upwards of 100 days to stabilize during batch (fill and draw) operation of subsurface wetlands and that the relative ratios of biomass components remain relatively constant during biofilm growth. Also, it appears that filtration of suspended solids results in biomass concentration at the inlet to the wetland.

Biofiltration as an odour abatement strategy.

The chemical, physical and biological processes occurring in biofiltration are reviewed. A survey of operating biofilter performances is also presented and includes some novel comparative methods. It is concluded that biofiltration is a simple and cost-effective technology for odour removal and that an understanding of the many interactions occuring within the biofilter is essential for the optimal performance of the biofilter.

Human tissue preservation for disaster victim identification (DVI) in tropical climates.

Disaster victim identification (DVI) poses unique challenges for forensic personnel. Typical scenarios may involve many bodies or body parts to identify in remote locations with limited access to laboratory facilities and in extreme temperatures. Transportation of tissue samples to a forensic laboratory for DNA profiling can take weeks without refrigeration. As well as protecting DNA for subsequent analysis, tissue preservation methods ideally should be safe, readily available and easy to transport to the scene at relatively low cost. We examined eight tissue preservatives (salt, DMSO, ethanol, ethanol with EDTA, TENT buffer, RNAlater(®), DNA Genotek Tissue Stabilising Kit and DNAgard(®)) and compared the quantity and quality of DNA recovered from human tissue and preservative solution stored at 35°C. Salt, DMSO, ethanol solutions, DNA Genotek and DNAgard(®) produced full Identifiler(®) genotypes up to one month from DNA extracts. In addition, DMSO, DNA Genotek and DNAgard(®) produced full profiles from aliquots of the liquid preservative.