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.

Duration of Ross River viraemia in a mouse model--implications for transfusion transmission.

BACKGROUND AND OBJECTIVES: There is little data on the duration of viraemia following infection with Ross River virus (RRV), the most common cause of arbovirus disease in Australia. In particular, no accurate estimate exists for the duration of pre-symptomatic RRV infection, which is important in assessing the potential for transfusion transmission. MATERIALS AND METHODS: We used an established mouse model of RRV infection involving adult Swiss outbred mice to measure viraemia following infection. Applying our experimental data to a published probabilistic model for estimating the risk of dengue transmission by transfused blood, we derived comparable risk estimates for RRV. RESULTS: Ross River virus RNA was measured using highly sensitive real-time PCR in serum samples to determine the duration of asymptomatic viraemia, which typically lasted 5 days, but extended to 9 days in some mice. Assuming the potential for transfusion transmission is proven, the risk of RRV transmission by blood during a 2004 outbreak in Cairns, Australia was retrospectively estimated as 1 in 13,542 (range from 1 in 4765 to 47,563). CONCLUSION: This study provides updated epidemiological data useful to underpin modelling to assess the potential risk of transfusion-transmitted RRV. Using an established model for dengue, the risk estimate for RRV transmission is comparable in the same geographical region. Should transfusion be proven as a route of transmission, this supports consideration of appropriate mitigation strategies to safeguard blood recipients.

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.

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.

Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction.

BACKGROUND: With decreased rates of HIV mortality and disease progression attributable to treatment with nucleoside analogue reverse transcriptase inhibitors (NRTIs), attention has now become focused on the toxicities of these forms of treatment. It is believed NRTIs cause a decrease in mitochondrial DNA (mtDNA) synthesis due to their inhibition of DNA polymerase gamma. This hypothesis is supported by in vitro data from muscle biopsies and human lymphoblastic cell lines. The resulting mitochondrial toxicity is thought to manifest itself in a variety of clinical symptoms including fatigue, fat wasting and peripheral neuropathy. A non-invasive test of mitochondrial toxicity is needed to assess toxicity and optimise HIV treatment strategies. Peripheral blood mononuclear cells (PBMC) and subcutaneous fat could be ideal and accessible sources of mtDNA for examining toxicity. OBJECTIVES: The objectives of this study were (a) to develop an assay to quantify the mtDNA copy number of PBMC and obtain reproducible results and (b) to establish the utility of subcutaneous fat as a source of mtDNA for quantification. STUDY DESIGN: PBMC were isolated from blood by centrifugation over Ficoll-Paque and subcutaneous fat was obtained from two 3 mm punch skin biopsies. Following DNA extraction, the mtDNA copy number in each sample was quantified by real-time polymerase chain reaction (PCR). RESULTS: The real-time PCR assay was found to generate consistent and reproducible results with replicates of samples undertaken within the same run, and in two or more different runs, having a mean coefficient of variation of 11.3 and 17.2%, respectively. PBMC and subcutaneous fat contained 409+/-148 and 2042+/-391 copies of mtDNA per cell, respectively. CONCLUSIONS: From the work carried out it can be concluded that firstly, the real-time PCR assay generates consistent and reproducible results, and secondly that mtDNA can be extracted and quantified from PBMC and subcutaneous fat.

Chlamydia pneumoniae in HIV-infected patients and controls assessed by a novel whole blood interferon-gamma assay, serology and PCR.

Chlamydia pneumoniae seropositivity is associated with cardiovascular disease and HIV infection. Cell-mediated immune responses are important for control of C. pneumoniae, and such responses may be impaired in HIV-infected patients. An assay for detection of interferon (IFN)-gamma in whole blood stimulated with C. pneumoniae antigen was developed and studied in HIV-infected patients and uninfected controls. Among 34 HIV-infected patients, none had an IFN-gamma response to C. pneumoniae antigen, compared with five of 32 healthy controls (p < 0.001). Fewer HIV-infected individuals elicited a serum IgG response when tested with a commercial enzyme immunoassay (p 0.009), but this was not so for serum IgA (p 0.12). Additionally, the IFN-gamma and antibody assays showed a trend towards a bivariate response in normal controls. This indicates that cellular and antibody responses against C. pneumoniae may be mutually exclusive, with potential implications for the role of this organism in the genesis of cardiovascular disease in both immunocompetent and HIV-infected populations.

Interleukin-15 mediates potent antiviral responses via an interferon-dependent mechanism.

Interleukin-15 (IL-15) is a potent growth factor for activated T and natural killer (NK) cells, stimulator of memory T cells and plays an important role in viral immunity. To investigate mechanisms underlying the antiviral activity of IL-15, a recombinant vaccinia virus (rVV) encoding murine IL-15 (VV-IL-15) was constructed. Following infection of mice with VV-IL-15, virus titres in the ovaries were significantly reduced compared to mice infected with control VV. Growth of VV-IL-15 was also reduced in nude athymic mice, indicating the antiviral activity of IL-15 does not require T cells. Additionally, VV-IL-15 augmented the cytolytic activity of natural NK cells in the spleen and enhanced interferon (IFN) mRNA expression and transcription factors associated with IFN induction. Using knockout mice and antibody depletion studies, we showed for the first time that the control of VV-IL-15 replication in mice is dependent on NK cells and IFNs and, in their absence, the protective role of IL-15 is abolished.