Journal Pre-proof J o u r n a l P r e -p r o o f 2 ABSTRACT Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections amongst the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.Journal Pre-proof de Roda Husman, 2020;Medema et al., 2020;Wu et al., 2020). Here, we report the first evidence for the presence of SARS-CoV-2 RNA in wastewater in Australia. Our preliminary findings demonstrate the applicability of WBE for COVID-19 surveillance as a potential tool for public health monitoring at the community level. Journal Pre-proof conditions ranging from 3 × 20 s at 8,000 rpm at a 10 s interval. From here on RNA was extracted using RNeasy Power Microbiome kit as per manufacturer's instruction. Method B began with centrifugation of wastewater samples (100-200 mL) at 4,750 g for 30 mins. Supernatant was then removed carefully without disturbing the pellet and Journal Pre-proof 4 /reaction) of Oncorhynchus keta (O. keta) was added in the DNAse and RNAse free water and the Cq value obtained acted as a reference point. If the Cq value of a wastewater sample increases compared to the reference Cq value, the sample is considered to have PCR inhibitors. Wastewater samples with a 2-Cq (quantification cycle) delay was considered to have RT-qPCR inhibition (Staley et al., 2012). All RNA samples were Journal Pre-proof average quality of 15 (SLIDINGWINDOW:4:15). Reads were cropped to 120bp (CROP:120), with any less than 120bp in length discarded (MINLEN:120). Overlapping forward and reverse reads were merged using bbmerge from the BBMap suite (ver. 38.41, https://sourceforge.net/projects/bbmap/). Quality-controlled, merged reads were then mapped Journal Pre-proof describing it, through the model 10,000 times. For each estimate of infected persons, the corresponding prevalence was calculated by dividing the number of persons infected by the number of persons in the catchment. Sensitivity of the estimated number o...

Marsupials exhibit great diversity in ecology and morphology. However, compared with their sister group, the placental mammals, our understanding of many aspects of marsupial evolution remains limited. We use 101 mitochondrial genomes and data from 26 nuclear loci to reconstruct a dated phylogeny including 97% of extant genera and 58% of modern marsupial species. This tree allows us to analyze the evolution of habitat preference and geographic distributions of marsupial species through time. We found a pattern of mesic-adapted lineages evolving to use more arid and open habitats, which is broadly consistent with regional climate and environmental change. However, contrary to the general trend, several lineages subsequently appear to have reverted from drier to more mesic habitats. Biogeographic reconstructions suggest that current views on the connectivity between Australia and New Guinea/Wallacea during the Miocene and Pliocene need to be revised. The antiquity of several endemic New Guinean clades strongly suggests a substantially older period of connection stretching back to the Middle Miocene and implies that New Guinea was colonized by multiple clades almost immediately after its principal formation.

The RNA modification N 6 -methyladenosine (m 6 A) influences mRNA stability and cell-type-specific developmental programming, and is highly abundant in the adult brain. However, it has not been determined whether m 6 A is dynamically regulated by experience.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus which causes COVID-19, has spread rapidly across the globe infecting millions of people and causing significant health and economic impacts. Authorities are exploring complimentary approaches to monitor this infectious disease at the community level. Wastewater-based Epidemiological (WBE) approaches to detect SARS-CoV-2 RNA in municipal wastewater are being developed worldwide as an environmental surveillance approach to inform health authority decision-making. Owing to the extended excretion of SARS-CoV-2 RNA in stool, WBE can surveil large populated areas with a longer detection window providing unique information on the presence of pre-symptomatic and asymptomatic cases that are unlikely to be screened by clinical testing. Herein, we analysed SARS-CoV-2 RNA in 24-h composite wastewater samples ( n = 63) from three wastewater treatment plants (WWTPs) in Brisbane, Queensland, Australia from 24 th of February to 1 st of May 2020. A total of 21 samples were positive for SARS-CoV-2, ranging from 135 to 11,992 gene copies (GC)/100 mL of wastewater. Detections were made in a Brisbane South WWTP in late February 2020, up to three weeks before the first cases were reported there. Wastewater samples were generally positive during the period with highest caseload data. The positive SARS-CoV-2 RNA detection in wastewater while there were limited clinical reported cases demonstrates the potential of WBE as an early warning system. When integrated into disease surveillance and monitoring systems, wastewater monitoring data may assist management efforts to identify hotspots and target localised public health responses, such as increased individual testing and the provision of health warnings.

Recent advances in next-generation sequencing technology allow high-throughput cDNA sequencing (RNA-Seq) to be widely applied in transcriptomic studies, in particular for detecting differentially expressed genes between groups. Many software packages have been developed for the identification of differentially expressed genes (DEGs) between treatment groups based on RNA-Seq data. However, there is a lack of consensus on how to approach an optimal study design and choice of suitable software for the analysis. In this comparative study we evaluate the performance of three of the most frequently used software tools: Cufflinks-Cuffdiff2, DESeq and edgeR. A number of important parameters of RNA-Seq technology were taken into consideration, including the number of replicates, sequencing depth, and balanced vs. unbalanced sequencing depth within and between groups. We benchmarked results relative to sets of DEGs identified through either quantitative RT-PCR or microarray. We observed that edgeR performs slightly better than DESeq and Cuffdiff2 in terms of the ability to uncover true positives. Overall, DESeq or taking the intersection of DEGs from two or more tools is recommended if the number of false positives is a major concern in the study. In other circumstances, edgeR is slightly preferable for differential expression analysis at the expense of potentially introducing more false positives.

BackgroundWe present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development.ResultsThe genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements.ConclusionsAnalyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Background Wastewater-based epidemiology (WBE) for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimise SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool. Methods Airline and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 RNA using two virus concentration methods, adsorption-extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative amplicons from positive samples were sequenced to confirm assay specificity. Results SARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however, concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimised false-negative results. Representative amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among assays and concentration methods. Conclusions The study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.

Cross-ethnic genetic studies can leverage power from differences in disease epidemiology and population-specific genetic architecture. In particular, the differences in linkage disequilibrium and allele frequency patterns across ethnic groups may increase gene-mapping resolution. Here we use cross-ethnic genetic data in sporadic amyotrophic lateral sclerosis (ALS), an adult-onset, rapidly progressing neurodegenerative disease. We report analyses of novel genome-wide association study data of 1,234 ALS cases and 2,850 controls. We find a significant association of rs10463311 spanning GPX3-TNIP1 with ALS (p = 1.3 × 10−8), with replication support from two independent Australian samples (combined 576 cases and 683 controls, p = 1.7 × 10−3). Both GPX3 and TNIP1 interact with other known ALS genes (SOD1 and OPTN, respectively). In addition, GGNBP2 was identified using gene-based analysis and summary statistics-based Mendelian randomization analysis, although further replication is needed to confirm this result. Our results increase our understanding of genetic aetiology of ALS.