PAG 2025

Targeted amplicon sequencing remains a standard approach in contexts ranging from biodiversity science to medical diagnostics. In some cases, many millions or even billions of samples require analysis, placing a premium on the development of protocols that are scalable and inexpensive. Short-read HTS lacks the ability to easily sequence longer amplicons, such as the 3819 bp gene encoding the spike protein of SARS-CoV-2 or even the 658 bp barcode region of the cytochrome c oxidase I gene used for species diagnosis. By contrast, these templates present no challenge to ONT. This presentation focuses on the capacity of ONT to speed the registration of the estimated 10 to 20 million species of multicellular organisms. Because of their lognormal abundance distribution, at least a billion specimens will require analysis to complete this task. Motivated by this fact, we assessed the capacity of ONT to support sequence recovery from amplicon pools which were previously sequenced on Sequel II (PacBio). When analysis was restricted to specimens represented by at least five reads, sequences generated by ONT were effectively identical to those from Sequel II. Moreover, recovery success matched that achieved by Sequel II, even when amplicons from 100,000 specimens were pooled and analyzed on a MinION, lowering the sequencing cost to just $0.01 each. Similarly high fidelity and recovery success was obtained when an amplicon pool from a million specimens was run on a single PromethION flow cell, lowering the sequencing cost by another order of magnitude. The potential application of this approach extends into medical contexts ranging from the characterization of the next pandemic agent or to the diagnostic evaluation of key marker genes as all eight billion humans could be analyzed for just $8 million. Potential applications extend into biodiversity writ large.

Species with temperature-dependent sex determination (TSD) face the risk of heavily skewed sex ratios under climate change, threatening population viability. Endangered sea turtles are particularly vulnerable, with many populations predicted to become entirely feminised by the century end. However, large-scale assessments of nest sex ratios are hindered by the lack of a reliable method for sexing hatchlings non-lethally, which must be rectified to guide conservation interventions as warming progresses. DNA methylation is an epigenetic mechanism involved in gene expression regulation, including of sex-related genes. High-quality genomic and epigenomic resources will therefore support efforts for identifying non-lethal biomarkers of sex in sea turtles. By leveraging Oxford Nanopore Technologies (ONT) sequencing, the Turtle Project simultaneously produced a chromosome-level genome assembly and methylome profile for loggerhead sea turtles (Caretta caretta) from the globally important Cabo Verde (Northeast Atlantic) rookery. We next performed epigenome-wide discovery scans that identified over 700 differentially methylated sites between the sexes of loggerhead hatchlings from minimally invasive blood samples. This set of sex-diagnostic molecular biomarkers offer a scalable solution that can now be applied to monitor sex ratios of wild sea turtle populations. Finally, we demonstrate the usage of an ONT P2 Solo platform as a portable tool for sequencing in the field, thus enabling the application of our biomarkers to inform real-time hatchery management decisions. This work represents a promising step towards a precision conservation approach for protecting the resilience of endangered species and populations under climate change.

Highly pathogenic avian influenza (HPAI) viruses are causing an ongoing panzootic in wild birds. Circulation of these viruses is associated with spillover infections in multiple species of mammals, including a large, unprecedented outbreak in American dairy cattle. Infected cattle can shed high amounts of HPAI H5N1 viruses in milk, allowing detection in pasteurized retail dairy samples. Over several months of sampling in one Midwestern city, we were able to obtain dairy products processed in 20 different states. Here we demonstrate a tiled-amplicon sequencing approach using the Oxford Nanopore Minion platform that produced over 90% genome coverage at greater than 20x depth from retail dairy samples. We also discuss findings and limitations of our method in the context of genomic surveillance on this rapidly-evolving outbreak. A combination of RT-qPCR testing and sequencing from retail dairy products can be a useful component of a One Health framework for responding to the avian influenza outbreak in cattle.

When you can assemble everything, then you can assemble anything. The dream of complete and accurate telomere-to-telomere (T2T) plant genome assemblies has finally become a reality. Recent advances in long-read sequencing technology have made this a relatively inexpensive and straightforward process, rather than a Herculean effort. T2T has become the new standard for plant reference genomes assemblies, yet T2T assemblies or approaches are not required for all plant genomes depending on the application. Thus, choosing when to pursue a T2T strategy has become a key consideration. However, the same methods that can be used to generate T2T assemblies for more cooperative genomes (as needed) can be used to tackle the most challenging plant genomes and genomic features as well.

De novo genome assembly forms the foundation of modern genomics research, providing the references against which genetic variation is mapped and recorded, as well as enabling complete, unbiased investigation of the genetic makeup of an organism or metagenomic community. Long reads are essential for complete and contiguous de novo assemblies as they can span repetitive regions and enable their accurate reconstruction. However, legacy long read assemblers do not fully leverage the accuracy and length of modern Oxford Nanopore reads, leading to sub-optimal performance and/or high compute costs. The new ont mode of the Hifiasm assembler overcomes these limitations, providing highly contiguous and complete genomes for inbred/haploid, diploid, and allotetraploid samples, all with a fraction of the compute requirements of legacy assemblers. Meanwhile, the new MetaMDBG assembler is able to reconstruct hundreds to thousands of high-quality metagenome assembled genomes (MAGs) in fecal and environmental samples. In this talk we will review these advances and the incredible assemblies they've produced, as well as discussing best-practices for plant, animal, and metagenomic de novo assembly experiments.