Macroinvertebrates and microbes (Archaea, Bacteria) offer complementary insights into mine-pit lake ecology

The broad objective of this research was to determine the environmental drivers of macroinvertebrate and microbial assemblages in acidic pit lakes. This is important because pit lake ecosystem development is influenced by prevailing environmental characteristics. Three lakes (Stockton, Kepwari, WO5H) within a larger pit-lake district in Collie, Western Australia were surveyed for spatial variability of benthic macroinvertebrate and microbe (Archaea, Bacteria) assemblage composition as well as potential environmental drivers (riparian condition, aquatic habitat, sediments, and aquatic chemistry) of assemblages. With the exception of sediment chemistry, biophysical variables were significantly different across lakes and reflected riparian condition and groundwater chemistry. Microbial assemblages in pit lakes were significantly different across lakes and correlated with water chemistry, particularly metals in Lake WO5H. However, the most abundant microbes were not readily identified beyond class, making it difficult to speculate on their ecological function. Macroinvertebrate assemblage composition and species richness were also significantly different across all lakes, and in Lake WO5H (a lake with low pH and high metal concentrations), taxa were correlated with benthic organic matter as well as water chemistry. Results indicated that despite poor water quality, input of nutrients from terrestrial leaf litter can support or augment pit lake ecosystems. This is a demonstration of the concept that connection of pit lakes to catchments can positively affect aquatic ecosystems, which can inform management actions for remediation

Detection of arboviruses and other micro-organisms in experimentally infected mosquitoes using massively parallel sequencing.

Human disease incidence attributed to arbovirus infection is increasing throughout the world, with effective control interventions limited by issues of sustainability, insecticide resistance and the lack of effective vaccines. Several promising control strategies are currently under development, such as the release of mosquitoes trans-infected with virus-blocking Wolbachia bacteria. Implementation of any control program is dependent on effective virus surveillance and a thorough understanding of virus-vector interactions. Massively parallel sequencing has enormous potential for providing comprehensive genomic information that can be used to assess many aspects of arbovirus ecology, as well as to evaluate novel control strategies. To demonstrate proof-of-principle, we analyzed Aedes aegypti or Aedes albopictus experimentally infected with dengue, yellow fever or chikungunya viruses. Random amplification was used to prepare sufficient template for sequencing on the Personal Genome Machine. Viral sequences were present in all infected mosquitoes. In addition, in most cases, we were also able to identify the mosquito species and mosquito micro-organisms, including the bacterial endosymbiont Wolbachia. Importantly, naturally occurring Wolbachia strains could be differentiated from strains that had been trans-infected into the mosquito. The method allowed us to assemble near full-length viral genomes and detect other micro-organisms without prior sequence knowledge, in a single reaction. This is a step toward the application of massively parallel sequencing as an arbovirus surveillance tool. It has the potential to provide insight into virus transmission dynamics, and has applicability to the post-release monitoring of Wolbachia in mosquito populations

Relationship of <i>Wolbachia</i> derived from <i>Ae. albopictus</i> and <i>Ae. aegypti</i> mosquitoes based on 16S ribosomal RNA sequence.

<p>Ribosomal RNA sequences were assembled using a reference sequence, and a multiple alignment file was constructed. This was used to calculate a distance matrix and phylogenetic tree using the neighbour joining method. For comparison, <i>Wolbachia</i> 16S ribosomal RNA from field-caught <i>Ae. albopictus</i> (Accession number X61767.1) and <i>D. melanogaster</i> (Accession number AE017196.1) were included, and 16S ribosomal RNA from <i>Rickettsia prowazekii</i> (Accession number NC_017560.1) was used as an out-group. The numbers shown are the bootstrapping values for 1000 replicates.</p

Mosquito and micro-organism ribosomal RNA analysis.

<p>1. The number of reads assigned by MEGAN to the LCA taxonomic level indicated: species (S), genus (G), family (F), order (O), phylum (P), or rankless taxon (T).</p><p>2. <i>Wolbachia</i> endosymbiont of <i>D. melanogaster</i>.</p><p>3. Trans-infected with <i>Wolbachia</i>.</p><p>4. Taxon without rank, <i>Coelomata</i>.</p

Detection of virus in experimentally-infected mosquitoes by MPS.

<p>1. Reference sequences (NCBI accession number): DENV-3, NC_001475.2; CHIKV, DQ443544.2; YFV, NC_002031.1.</p><p>2. Detection by Taqman assay – threshold value (C_t).</p><p>3. For DENV-3, CHIKV and YFV, values were the number of reads matched using the reference assembly feature of GeneiousPro. For densovirus and inovirus, values were the number of reads matched by standalone BLASTn followed by MEGAN.</p><p>4. Not applicable.</p

Comparison of sequence coverage and depth of the virus genomes.

<p>Products were generated using sequence-independent amplification from mosquitoes 1 and 2, which were infected with DENV-3 (A and B, respectively), mosquitoes 2 and 3, which were infected with CHIKV (C and D, respectively), and mosquito 5, which was infected with YFV and amplified either using sequence-independent amplification (E) or GenomiPhi amplification (F). These products were sequenced and assembled to a reference sequence as shown below the coverage diagram for each sample (DENV-3, NC_001475.2; CHIKV, DQ443544.2; YFV, NC_002031.1). The genome coverage (x-axis) and depth of coverage (y-axis) is shown.</p

Sequencing of the Viral UL111a Gene Directly from Clinical Specimens Reveals Variants of HCMV-Encoded IL-10 That Are Associated with Altered Immune Responses to HCMV

Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ~80% of adults worldwide. Acute infections are often asymptomatic in healthy individuals but generate diverse syndromes in neonates, renal transplant recipients (RTR), and people with HIV (PWH). The HCMV gene UL111a encodes a homolog of human interleukin-10 (IL-10) that interacts with the human IL-10 receptor. Deep sequencing technologies were used to sequence UL111a directly from 59 clinical samples from Indonesian PWH and Australian RTR, healthy adults, and neonates. Overall, 93% of samples contained more than one variant of HCMV, as defined by at least one nonsynonymous variation. Carriage of these variants differed between neonates and adults, Australians and Indonesians, and between saliva and blood leukocytes. The variant alleles of N41D and S71Y occurred together in Australian RTR and were associated with higher T-cell responses to HCMV pp65. The variant P122S was associated with lower levels of antibodies reactive with a lysate of HCMV-infected fibroblasts. L174F was associated with increased levels of antibodies reactive with HCMV lysate, immediate-early 1 (IE-1), and glycoprotein B (gB) in Australian RTR and Indonesians PWH, suggesting a higher viral burden. We conclude that variants of UL111a are common in all populations and may influence systemic responses to HCMV

Targeted gene panel use in 2249 neuromuscular patients: the Australasian referral center experience.

To develop, test, and iterate a comprehensive neuromuscular targeted gene panel in a national referral center. We designed two iterations of a comprehensive targeted gene panel for neuromuscular disorders. Version 1 included 336 genes, which was increased to 464 genes in Version 2. Both panels used TargetSeqTM probe-based hybridization for target enrichment followed by Ion Torrent sequencing. Targeted high-coverage sequencing and analysis was performed on 2249 neurology patients from Australia and New Zealand (1054 Version 1, 1195 Version 2) from 2012 to 2015. No selection criteria were used other than referral from a suitable medical specialist (e.g., neurologist or clinical geneticist). Patients were classified into 15 clinical categories based on the clinical diagnosis from the referring clinician. Six hundred and sixty-five patients received a genetic diagnosis (30%). Diagnosed patients were significantly younger that undiagnosed patients (26.4 and 32.5 years, respectively; P = 4.6326E-9). The diagnostic success varied markedly between disease categories. Pathogenic variants in 10 genes explained 38% of the disease burden. Unexpected phenotypic expansions were discovered in multiple cases. Triage of unsolved cases for research exome testing led to the discovery of six new disease genes. A comprehensive targeted diagnostic panel was an effective method for neuromuscular disease diagnosis within the context of an Australasian referral center. Use of smaller disease-specific panels would have precluded diagnosis in many patients and increased cost. Analysis through a centralized laboratory facilitated detection of recurrent, but under-recognized pathogenic variants

Targeted gene panel use in 2249 neuromuscular patients: the Australasian referral center experience

[Objective] To develop, test, and iterate a comprehensive neuromuscular targeted gene panel in a national referral center.[Methods] We designed two iterations of a comprehensive targeted gene panel for neuromuscular disorders. Version 1 included 336 genes, which was increased to 464 genes in Version 2. Both panels used TargetSeqTM probe‐based hybridization for target enrichment followed by Ion Torrent sequencing. Targeted high‐coverage sequencing and analysis was performed on 2249 neurology patients from Australia and New Zealand (1054 Version 1, 1195 Version 2) from 2012 to 2015. No selection criteria were used other than referral from a suitable medical specialist (e.g., neurologist or clinical geneticist). Patients were classified into 15 clinical categories based on the clinical diagnosis from the referring clinician.[Results] Six hundred and sixty‐five patients received a genetic diagnosis (30%). Diagnosed patients were significantly younger that undiagnosed patients (26.4 and 32.5 years, respectively; P = 4.6326E‐9). The diagnostic success varied markedly between disease categories. Pathogenic variants in 10 genes explained 38% of the disease burden. Unexpected phenotypic expansions were discovered in multiple cases. Triage of unsolved cases for research exome testing led to the discovery of six new disease genes.[Interpretation] A comprehensive targeted diagnostic panel was an effective method for neuromuscular disease diagnosis within the context of an Australasian referral center. Use of smaller disease‐specific panels would have precluded diagnosis in many patients and increased cost. Analysis through a centralized laboratory facilitated detection of recurrent, but under‐recognized pathogenic variants.Research funding: The Fred Liuzzi Foundation, Australian Postgraduate Award, Australian Genomics Health Alliance. Grant Number: GNT1113531, Fundación Alfonso Martín Escudero, Junta de Andalucía‐Consejería de Salud. Grant Number: B‐0005‐2017, Australian National Health and Medical Research Council. Grant Numbers: APP1117510, APP1122952, APP1080587, Western Australian Department of Health Future Health’s WA Merit Award