Biology subjects

Rockett, R. J.

Publications and source records attributed to Rockett, R. J..

9 recordsLinked to original sources

bioRxiv2026

COSMOS: integrated vector-pathogen metagenomics for mosquito-borne disease

By 2050, almost half of the global population will be at risk of mosquito-borne disease. Effective response requires monitoring both pathogen transmission and vector abundance, yet these are typically assessed using separate assays with limited taxonomic breadth. Here we present COSMOS (COmprehensive Sequencing for MOSquito-borne disease), a targeted metagenomics workflow designed to reconstruct pathogen genomes and mosquito community composition directly from mixed field-collected samples. Over three annual rounds of a major Australian surveillance program, we evaluated >120,000 mosquitoes in 219 traps, with 98.7% agreement with surveillance data across seven monitored arboviruses, and additional detections of Sindbis virus and the state's first detection of encephalitic Little Sussex virus. Of 137 viral detections, 101 yielded near-complete genomes (>90% coverage) at median 95x depth. Using an accompanying computational workflow, COSMOS quantified mosquito composition, matching entomological estimates of reported vector species (CCC 0.78-0.89). Joint analysis of virus and vector composition identified structured mosquito communities associated with spatial and temporal environmental variation, allowing the ecological context of arbovirus detections to be characterised. Detections during the Murray Valley encephalitis virus outbreak in 2023 were concentrated within a narrowly defined mosquito-community space corresponding to rural communities dominated by the inland S-AUS sub-lineage of Culex annulirostris. Integrated vector-pathogen metagenomics provides ecological and evolutionary context for arbovirus activity within a single scalable workflow for surveillance and outbreak response.

microbiology↗

bioRxiv2026

Differential sensitivity of k-mer based identification tools using closely related database sequences affects relative abundance estimation

Deep metagenomic sequencing is increasingly used for lineage profiling of complex biological mixtures, including applications such as wastewater virus surveillance and microbiome profiling. For profiling, sequence read sets are compared against a database of reference sequences to identify component genetic lineages, and estimate their relative abundance. However, there is currently a lack of clarity regarding the impact of two ubiquitous features of biological mixtures on compositional profiling: (1) population diversity, where reads contain genetic diversity not represented in the reference set, and (2) the presence of high sequence similarity between references, which causes ambiguity in read assignment. Here we show, using both simulated and real data, that performance of two commonly used tools for lineage profiling, KMA and Kallisto, is differently affected by sample-reference distance and the presence of highly similar competing reference sequences. Increasing sample-reference distance reduced read assignment for both tools, but more strongly for Kallisto, which led to directional bias in abundance estimates when distance differed between mixture components. In contrast, when sample-reference distance was low, Kallisto more specifically discriminated between highly similar competing references, whereas KMA was less able to partition reads accurately between them. This specificity-sensitivity trade-off was reflected in real datasets: KMA produced more accurate abundance estimates for mosquito COX1 mixtures, whereas Kallisto performed better for mixtures of closely related SARS-CoV-2 lineages, although neither tool fully resolved the most similar SARS-CoV-2 variants. Tool choice is therefore dependent on the degree of expected sample-reference distance and inter-reference distance within datasets.

bioinformatics↗

bioRxiv2023

Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine

AimsTo explore viral evolution during in vitro neutralisation using next generation sequencing, and to determine whether sera from individuals immunised with two doses of the Pfizer-BioNTech vaccine (BNT162b2) are as effective at neutralising the SARS-CoV-2 variant of concern (VOC) Delta (B 1.617.2) compared to the earlier lineages Beta (B.1.351) and wild-type (A.2.2) virus. MethodsUsing a live-virus SARS-CoV-2 neutralisation assay in Vero E6 cells we determined neutralising antibody titres (nAbT) in 14 participants (vaccine-naive (n=2) and post-second dose of BNT162b2 vaccination (n=12), median age 45 years [IQR 29-65], median time after second dose = 21 days [IQR 19-28] against three SARS-CoV-2 strains: wild-type, Beta and Delta. The determination of nAbT was performed by visual inspection of cytopathic effect (CPE) and in-house quantitative reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR) to confirm SARS-CoV-2 replication. A total of 110 representative samples including inoculum, neutralisation breakpoints at 72 hrs, negative and positive controls underwent genome sequencing using the Respiratory Viral Oligo Panel version 2 (RVOP) (Illumina Inc. (San Diego, United States of America)) viral enrichment and short read sequencing using (Illumina Inc.San Diego, United States of America)(Figure 1). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=35 SRC="FIGDIR/small/558921v1_fig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1f04650org.highwire.dtl.DTLVardef@1986fceorg.highwire.dtl.DTLVardef@d47ab9org.highwire.dtl.DTLVardef@1c524d5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Outline of virus neutralisation assays of emerging SARS-CoV-2 variants of concern C_FIG ResultsThere was a significant reduction in nAbT observed against the Delta and Beta VOC compared with wild-type, 4.4-fold (p = >0.0006) and 2.3-fold (p = 0.0140), respectively (Figure 2). Neutralizing antibodies were not detected in one vaccinated immunosuppressed participant nor the vaccine-naive participants (n=2). The highest nAbT against the SARS-CoV-2 variants investigated was obtained from a participant who was vaccinated following SARS-CoV-2 infection 12 months prior (Table S1). Limited consensus level mutations occurred in the SARS-CoV-2 genome of any lineage during in vitro neutralisation, however, consistent minority allele frequency variants (MFV) were detected in the SARS-CoV-2 polypeptide, spike (S) and membrane protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/558921v1_fig2.gif" ALT="Figure 2"> View larger version (18K): org.highwire.dtl.DTLVardef@1f51ee8org.highwire.dtl.DTLVardef@1c03b61org.highwire.dtl.DTLVardef@17a77d2org.highwire.dtl.DTLVardef@15084f4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2:C_FLOATNO Neutralising Antibody Titres Figure description: Differences in neutralising antibody titres (nAbT) against the Wildtype, Beta and Delta SARS-CoV-2 lineages median of 21 days (IQR 19-28) after receiving the second dose of Pfizer-BioNTech (BNT162b2) vaccine measured by visual inspection of cytopathic effect (CPE) with confirmation of viral replication by SARS-CoV-2 by inhouse reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR). Results are reported in the box-whiskers plots as medians and upper and lower quartiles. There was a significant fold reduction in nAbT observed between both the Delta (M = 4.4, SD = 2), t(11) =-4.9, p = .00059, and Beta (M=2.3, SD=2) t(11) =-3 p = .01397 compared with wild-type. There was also a significant fold reduction in nAbT between Beta (M = 2.6, SD = 1.4), t(11) =-2.5, p = .02897 and Delta. Key: Delta - Delta (B.1.617.2) lineage; Beta - Beta (B.1.351) lineage; Wildtype - Wildtype (A.2.2) lineage; C_FIG DiscussionSignificant reductions in nAbT post-vaccination were identified, with Delta demonstrating a 4.4-fold reduction. The reduction in nAbT for the VOC Beta has been previously documented, however, limited data is available on vaccine evasion for the Delta VOC, the predominant strain currently circulating worldwide at the time. Studies in high incidence countries may not be applicable to low incidence settings such as Australia as nAbT may be significantly higher in vaccine recipients previously infected with SARS-CoV-2, as seen in our cohort. Monitoring viral evolution is critical to evaluate the impact of novel SARS-CoV-2 variants on vaccine effectiveness as mutational profiles in the sub-consensus genome could indicate increases in transmissibility, virulence or allow the development of antiviral resistance.

microbiology↗

bioRxiv2023

A laboratory framework for ongoing optimisation of amplification based genomic surveillance programs

Constantly evolving viral populations affect the specificity of primers and quality of genomic surveillance. This study presents a framework for continuous optimisation of sequencing efficiency for public health surveillance based on the ongoing evolution of the COVID-19 pandemic. SARS-CoV-2 genomic clustering capacity based on three amplification based whole genome sequencing schemes was assessed using decreasing thresholds of genome coverage and measured against epidemiologically linked cases. Overall genome coverage depth and individual amplicon depth were used to calculate an amplification efficiency metric. Significant loss of genome coverage over time was documented which was recovered by optimisation of primer pooling or implementation of new primer sets. A minimum of 95% genome coverage was required to cluster 94% of epidemiologically defined SARS-CoV-2 transmission events. Clustering resolution fell to 70% when only 85% of genome coverage was achieved. The framework presented in this study can provide public health genomic surveillance programs a systematic process to ensure an agile and effective laboratory response during rapidly evolving viral outbreaks.

genomics↗

bioRxiv2021

Improved neutralization of the SARS-CoV-2 Omicron variant after Pfizer-BioNTech BNT162b2 COVID-19 vaccine boosting

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired 15 mutations in the receptor binding domain of the spike protein, raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three and six months post-two doses of Pfizer-BioNTech BNT162b2 has a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres are boosted. Despite this increase, neutralising antibody titres are reduced four-fold for Omicron compared to lineage A.2.2 SARS-CoV-2.

microbiology↗

bioRxiv2021

SARS-CoV-2 Genome Sequencing Methods Differ In Their Ability To Detect Variants From Low Viral Load Samples

SARS-CoV-2 genomic surveillance has been vital in understanding the spread of COVID-19, the emergence of viral escape mutants and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low frequency variants. Our analyses highlight that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina respiratory viral oligo panel, and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high quality sequence data from low viral load samples for public health and genomic surveillance purposes.

microbiology↗

bioRxiv2020

REVEALING COVID-19 TRANSMISSION BY SARS-CoV-2 GENOME SEQUENCING AND AGENT BASED MODELLING

Community transmission of the new coronavirus SARS-CoV-2 is a major public health concern that remains difficult to assess. We present a genomic survey of SARS-CoV-2 from a during the first 10 weeks of COVID-19 activity in New South Wales, Australia. Transmission events were monitored prospectively during the critical period of implementation of national control measures. SARS-CoV-2 genomes were sequenced from 209 patients diagnosed with COVID-19 infection between January and March 2020. Only a quarter of cases appeared to be locally acquired and genomic-based estimates of local transmission rates were concordant with predictions from a computational agent-based model. This convergent assessment indicates that genome sequencing provides key information to inform public health action and has improved our understanding of the COVID-19 evolution from outbreak to epidemic.

microbiology↗

bioRxiv2020

Genomic surveillance enables suitability assessment of Salmonella gene targets used for culture-independent diagnostic testing

Salmonella is a highly diverse genus consisting of over 2600 serovars responsible for high-burden food- and water-borne gastroenteritis worldwide. Sensitivity and specificity of PCR-based culture-independent diagnostic testing (CIDT) systems for Salmonella, which depend on a highly conserved gene target, can be affected by single nucleotide polymorphisms (SNPs), indels and genomic rearrangements within primer and probe sequences. This report demonstrates the value of prospectively collected genomic data for verifying CIDT targets. We utilised the genomes of 3165 Salmonella isolates prospectively collected and sequenced in Australia. The sequence of Salmonella CIDT PCR gene targets (ttrA, spaO and invA) were systematically interrogated to measure nucleotide dissimilarity. Analysis of 52 different serovars and 79 MLST types demonstrated dissimilarity within and between PCR gene targets ranging between 0 - 81.3 SNP/Kbp (0 and 141 SNPs). Lowest average dissimilarity was observed in the ttrA target gene used by the Roche LightMix at 2.0 SNP/Kbp [range 0 - 46.7]), however entropy across the gene demonstrates it may not be the most stable CIDT target. While debate continues over the benefits and pitfalls of replacing bacterial culture with molecular assays, the growing volumes of genomic surveillance data enable periodic regional reassessment and validation of CIDT targets against both prevalent and emerging serovars. If PCR systems are to become the primary screening and diagnostic tool for laboratory diagnosis of salmonellosis, ongoing monitoring of the genomic diversity in PCR target regions is warranted as is the potential inclusion of two Salmonella PCR targets into frontline diagnostic systems.

microbiology↗

bioRxiv2019

Complete microbial genomes for public health in Australia and Southwest Pacific

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health lab activities. Here, we present complete genomes of pathogen strains of regional importance to the Southwest Pacific and Australia. These enrich the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health labs.\n\nAnnouncementWhole-genome sequence (WGS) data is increasingly important in public health microbiology (1-4). The data can be used to replicate many of the basic bacterial sub-typing approaches, as well as support epidemiological investigations, such as surveillance and outbreak investigation (5-7). The appeal of WGS data comes from the promise of a single workflow to process all microbial pathogens that can provide easily portable data that promotes deeper integration of surveillance and investigation efforts across jurisdictions. This promise is leading to a concerted effort to move microbial public health to a primarily genome-based workflow at numerous jurisdictions (8-10), including Australia (11).

genomics↗