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ABSTRACT Highly pathogenic avian influenza viruses continue to pose global risks to One Health, including agriculture, public, and animal health. Rapid and accurate genomic surveillance is critical for monitoring viral mutations, tracing transmission, and guiding interventions in near real-time. Oxford Nanopore sequencing holds promise for real-time influenza genotyping, but data quality from R9 chemistry has limited its adoption due to challenges resolving low-complexity regions such as the biologically critical hemagglutinin cleavage site, a homopolymer of basic amino acids that distinguish highly pathogenic strains. In this study, human and avian influenza isolates (n = 45) from Cambodia were sequenced using both R9.4.1 and R10.4.1 flow cells and chemistries to evaluate performance between approaches. Overall, R10.4.1 yielded increased data output with higher average quality compared to R9.4.1, producing improved consensus sequences using a reference-based bioinformatics approach. R10.4.1 had significantly lower minor population insertion and deletion frequencies, driven by improved performance in low sequence complexity regions prone to insertion and deletion errors, such as homopolymers. Within the hemagglutinin cleavage site, R10.4.1 resolved the correct motif in 90% of genomes compared to only 60% with R9.4.1. Further examination showed reduced frameshift mutations in consensus sequences generated with R10.4.1 that could result in incorrectly classified virulence with automated pipelines. Improved consensus genome quality from nanopore sequencing approaches, especially across biologically important low-complexity regions, is critical to reduce subjective hand-curation and will improve local and global genomic surveillance responses.IMPORTANCEThis study demonstrates significant advancement in the field of influenza virus genomic surveillance by showcasing the superior accuracy and data quality of the Oxford Nanopore R10 sequencing chemistry compared to the older R9 chemistry. Improved resolution, including in the critical hemagglutinin multi-basic cleavage site, enables more reliable monitoring and tracking of viral mutations. This accelerates the ability to respond quickly to outbreaks, potentially improving impacts on public health, agriculture, and the economy by enabling more accurate and timely interventions.

Abstract Background COVID-19 waves caused by specific SARS-CoV-2 variants have occurred globally at different times. We focused on Omicron variants to understand the genomic diversity and phylogenetic relatedness of SARS-CoV-2 strains in various regions of Pakistan. Methods We studied 276,525 COVID-19 cases and 1,031 genomes sequenced from December 2021 to August 2022. Sequences were analyzed and visualized using phylogenetic trees. Results The highest case numbers and deaths were recorded in Sindh and Punjab, the most populous provinces in Pakistan. Omicron variants comprised 93% of all genomes, with BA.2 (32.6%) and BA.5 (38.4%) predominating. The first Omicron wave was associated with the sequential identification of BA.1 in Sindh, then Islamabad Capital Territory, Punjab, Khyber Pakhtunkhwa (KP), Azad Jammu Kashmir (AJK), Gilgit-Baltistan (GB) and Balochistan. Phylogenetic analysis revealed Sindh to be the source of BA.1 and BA.2 introductions into Punjab and Balochistan during early 2022. BA.4 was first introduced in AJK and BA.5 in Punjab. Most recent common ancestor (MRCA) analysis revealed relatedness between the earliest BA.1 genome from Sindh with Balochistan, AJK, Punjab and ICT, and that of first BA.1 from Punjab with strains from KPK and GB. Conclusions Phylogenetic analysis provides insights into the introduction and transmission dynamics of the Omicron variant in Pakistan, identifying Sindh as a hotspot for viral dissemination. Such data linked with public health efforts can help limit surges of new infections.

Abstract The economic and methodological efficiencies of environmental DNA (eDNA) based survey approaches provide an unprecedented opportunity to assess and monitor aquatic environments. However, instances of inadequate communication from the scientific community about confidence levels, knowledge gaps, reliability, and appropriate parameters of eDNA‐based methods have hindered their uptake in environmental monitoring programs and, in some cases, has created misperceptions or doubts in the management community. To help remedy this situation, scientists convened a session at the Second National Marine eDNA Workshop to discuss strategies for improving communications with managers. These include articulating the readiness of different eDNA applications, highlighting the strengths and limitations of eDNA tools for various applications or use cases, communicating uncertainties associated with specified uses transparently, and avoiding the exaggeration of exploratory and preliminary findings. Several key messages regarding implementation, limitations, and relationship to existing methods were prioritized. To be inclusive of the diverse managers, practitioners, and researchers, we and the other workshop participants propose the development of communication workflow plans, using RACI (Responsible, Accountable, Consulted, Informed) charts to clarify the roles of all pertinent individuals and parties and to minimize the chance for miscommunications. We also propose developing decision support tools such as Structured Decision‐Making (SDM) to help balance the benefits of eDNA sampling with the inherent uncertainty, and developing an eDNA readiness scale to articulate the technological readiness of eDNA approaches for specific applications. These strategies will increase clarity and consistency regarding our understanding of the utility of eDNA‐based methods, improve transparency, foster a common vision for confidently applying eDNA approaches, and enhance their benefit to the monitoring and assessment community.

Abstract Environmental DNA (eDNA) data make it possible to measure and monitor biodiversity at unprecedented resolution and scale. As use‐cases multiply and scientific consensus grows regarding the value of eDNA analysis, public agencies have an opportunity to decide how and where eDNA data fit into their mandates. Within the United States, many federal and state agencies are individually using eDNA data in various applications and developing relevant scientific expertise. A national strategy for eDNA implementation would capitalize on recent scientific developments, providing a common set of next‐generation tools for natural resource management and public health protection. Such a strategy would avoid patchwork and possibly inconsistent guidelines in different agencies, smoothing the way for efficient uptake of eDNA data in management. Because eDNA analysis is already in widespread use in both ocean and freshwater settings, we focus here on applications in these environments. However, we foresee the broad adoption of eDNA analysis to meet many resource management issues across the nation because the same tools have immediate terrestrial and aerial applications.

Setaria viridis (green foxtail) is an important model system for improving cereal crops due to its diploid genome, ease of cultivation, and use of C4 photosynthesis. The S. viridis accession ME034V is exceptionally transformable, but the lack of a sequenced genome for this accession has limited its utility. We present a 397 Mb highly contiguous de novo assembly of ME034V using ultra-long nanopore sequencing technology (read N50 = 41kb). We estimate that this genome is largely complete based on our updated k-mer based genome size estimate of 401 Mb for S. viridis. Genome annotation identified 37,908 protein-coding genes and >300k repetitive elements comprising 46% of the genome. We compared the ME034V assembly with two other previously sequenced Setaria genomes as well as to a diversity panel of 235 S. viridis accessions. We found the genome assemblies to be largely syntenic, but numerous unique polymorphic structural variants were discovered. Several ME034V deletions may be associated with recent retrotransposition of copia and gypsy LTR repeat families, as evidenced by their low genotype frequencies in the sampled population. Lastly, we performed a phylogenomic analysis to identify gene families that have expanded in Setaria, including those involved in specialized metabolism and plant defense response. The high continuity of the ME034V genome assembly validates the utility of ultra-long DNA sequencing to improve genetic resources for emerging model organisms. Structural variation present in Setaria illustrates the importance of obtaining the proper genome reference for genetic experiments. Thus, we anticipate that the ME034V genome will be of significant utility for the Setaria research community.

The early COVID-19 pandemic was characterized by rapid global spread. In Maryland and Washington, DC, United States, more than 2500 cases were reported within 3 weeks of the first COVID-19 detection in March 2020. We aimed to use genomic sequencing to understand the initial spread of SARS-CoV-2 — the virus that causes COVID-19 — in the region. We analyzed 620 samples collected from the Johns Hopkins Health System during March 11–31, 2020, comprising 28.6% of the total cases in Maryland and Washington, DC. From these samples, we generated 114 complete viral genomes. Analysis of these genomes alongside a subsampling of over 1000 previously published sequences showed that the diversity in this region rivaled global SARS-CoV-2 genetic diversity at that time and that the sequences belong to all of the major globally circulating lineages, suggesting multiple introductions into the region. We also analyzed these regional SARS-CoV-2 genomes alongside detailed clinical metadata and found that clinically severe cases had viral genomes belonging to all major viral lineages. We conclude that efforts to control local spread of the virus were likely confounded by the number of introductions into the region early in the epidemic and the interconnectedness of the region as a whole.

In 2010, the Conference of the Parties of the Convention on Biological Diversity agreed on the Strategic Plan for Biodiversity 2011–2020 in Aichi Prefecture, Japan. As this plan approaches its end, we discussed whether marine biodiversity and prediction studies were nearing the Aichi Targets during the 4th World Conference on Marine Biodiversity held in Montreal, Canada in June 2018. This article summarises the outcome of a five-day group discussion on how global marine biodiversity studies should be focused further to better understand the patterns of biodiversity. We discussed and reviewed seven fundamental biodiversity priorities related to nine Aichi Targets focusing on global biodiversity discovery and predictions to improve and enhance biodiversity data standards (quantity and quality), tools and techniques, spatial and temporal scale framing, and stewardship and dissemination. We discuss how identifying biodiversity knowledge gaps and promoting efforts have and will reduce such gaps, including via the use of new databases, tools and technology, and how these resources could be improved in the future. The group recognised significant progress toward Target 19 in relation to scientific knowledge, but negligible progress with regard to Targets 6 to 13 which aimed to safeguard and reduce human impacts on biodiversity.