Matthew Loose

Lateral gene transfer (LGT) from organellar to eukaryotic genomes is ubiquitous, resulting in the presence of widespread nuclear mitochondrial DNA segments (NUMTs) and/or plastid gene transfers. While LGT from bacterial associates of eukaryotes is generally less common, many arthropod genomes are littered with LGT originating from Wolbachia (order Rickettsiales) - a vertically transmitted, obligate intracellular symbiont of invertebrates. This contrasts with Rickettsia , a related genus including many important human pathogens, which has not been shown to promulgate LGT within its arthropod vectors. Here, we present the 8.6 Gb genome of a cell line derived from Amblyomma variegatum (the tropical bont tick), which is the primary vector of Rickettsia africae (agent of African tick-bite fever). In addition to numerous NUMTs and diverse families of transposable elements, an almost-complete chromosome of R. africae origin was identified in the cell line genome, localised to the putative sex chromosome. Sequencing of field-collected A. variegatum confirmed the presence of this large LGT in wild ticks, although the absence of transferred genes from the R. africae plasmid provided a means to differentiate LGT from genuine rickettsial infections. These findings highlight the imperative to consider the possibility of LGT when screening vectors for pathogens by PCR.

Background: Chromosome-level de novo genome assemblies are vital for many aspects of biological research. Despite technological progress, achieving telomere-to-telomere assemblies remains challenging. Long-read data are critical for a contiguous genome assembly, since repeat regions cannot be assembled without them. Researchers typically generate substantial quantities of data from multiple sequencing platforms to achieve accurate and contiguous telomere-to-telomere (T2T) assemblies. This makes the process costly and complicated. Recent advances in algorithms designed to optimise assembly with long-read data have the potential to rectify this issue. Using only Oxford Nanopore Technology (ONT) simplex long-read data from the Sumatran tiger ( Panthera tigris sumatrae ), we evaluate leading methods for error correction of long-reads (NextDenovo, HERRO and hifiasm ONT) and various assembly approaches. Results: We show that correcting errors in ONT long-reads during assembly greatly improves the quality and contiguity of the resulting assembly, suggesting these methods will make high quality genome assemblies more achievable with less data. We also present the first, almost complete, T2T, de novo genome assembly for the Sumatran tiger, with a single technology. Conclusions: This assembly is a novel resource for genomic research and conservation efforts.

Prior to the emergence of SARS-CoV-2 in 2019, alphacoronaviruses 229E and NL63 and betacoronaviruses OC43 and HKU1 were already established endemic ‘common cold’ viral infections. Despite their collective contribution towards global respiratory morbidity and mortality and potential to inform the future trajectory of SARS-CoV-2 endemicity, they are infrequently sequenced. We therefore developed a 1,200 bp amplicon whole-genome sequencing scheme targeting all four seasonal coronaviruses and SARS-CoV-2. The ‘Vivaldi’ method was applied retrospectively and prospectively using Oxford Nanopore Technology to ~400 seasonal coronavirus infections diagnosed in Nottingham, UK, from February 2016 to July 2023. We demonstrate that the amplicon multiplex strategy can be applied agnostically to determine the complete genomes of five different species from two coronaviral genera. Three hundred and four unique seasonal coronavirus genomes of greater than 95% coverage were achieved: 64 for 229E, 85 for NL63, 128 for OC43 and 27 for HKU1. They collectively indicated a dynamic seasonal coronavirus genomic landscape, with co-circulation of multiple variants emerging and declining over the UK winter respiratory infection season, with further geographical distinction when compared to a global dataset. Prolonged infection with concomitant intra-host evolution was also observed for both alpha- (NL63) and betacoronaviruses (OC43). This data represents the largest single cohort of seasonal coronavirus genomes to date and also a novel amplicon scheme for their future global surveillance suitable for widespread and easy adoption in the post-SARS-CoV-2 era of viral genomics.

Background Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections (LTRIs) in young children and adults over 65, contributing significantly to global healthcare burdens. With the recent approval of multiple pharmacological interventions for RSV, there is an increased demand for efficient, high-throughput sequencing methods to monitor RSV genetic diversity and any potential impact these interventions may have. Method To address this, we introduce two novel amplicon-based sequencing schemes designed for RSV A and B, optimised for integration with widespread existing ARTIC sequencing workflows. To examine their effectiveness at sequencing RSV samples from across the globe, eight external laboratories used these primers in their sequencing workflows, using both Illumina and Nanopore sequencing technologies. Results We demonstrate that these primer schemes can produce high quality genomes from a diverse range of RSV samples, with eight laboratories in five countries generating complete genomes on both Nanopore and Illumina sequencing platforms. The ability to effectively multiplex these RSV A and B primer schemes, enables streamlined, high-throughput sequencing without prior subtyping. Furthermore, these results provide a snapshot of the circulating diversity of RSV. Phylogenetic analysis of the 882 samples sequenced for this study suggests only minimal geographic clustering of RSV sequences, underscoring the global nature of RSV spread. It also highlights the distinct lineage dynamics seen between RSV A and B. Conclusions This study represents an advancement in RSV genomics, providing robust tools for global sequencing efforts aimed at tracking RSV evolution and assessing the efficacy of new therapeutic interventions both rapidly and at scale.

Background: Advances in our technological capacity to interrogate CNS tumor biology have led to the ever increasing use of genomic sequencing in diagnostic decision making. Presently, CNS tumors are classified based on their epigenetic signatures, leading to a paradigm shift in diagnostic pathways. Such testing can be performed so rapidly using nanopore sequencing that results can be provided intraoperatively. This information greatly improves the fidelity of smear diagnosis and can help surgeons tailor their approach, balancing the risks of surgery with the likely benefit. Nevertheless, full integrated diagnosis may require subsequent additional assays to detect pathognomonic somatic mutations and structural variants, thereby delaying the time to final diagnosis. Methods: Here, we present ROBIN, a tool based on PromethION nanopore sequencing technology that can provide both real-time, intraoperative methylome classification and next-day comprehensive molecular profiling within a single assay. ROBIN utilizes 3 methylation classifiers to improve diagnostic performance in the intraoperative setting. Results: We demonstrate classifier performance on 50 prospective intraoperative cases, achieving a diagnostic turnaround time under 2 hours and generating robust tumor classifications within minutes of sequencing. Furthermore, ROBIN can detect single nucleotide variants, copy number variants, and structural variants in real time, and is able to inform a complete integrated diagnosis within 24 hours. Classifier performance demonstrated concordance with final integrated diagnosis in 90% of prospective cases. Conclusion: Nanopore sequencing can greatly improve turnaround times for standard-of-care diagnostic testing and is furthermore able to reliably provide clinically actionable intraoperative tumor classification.

Since the advent of long-read sequencing, achieving longer read lengths has been a key goal for many users. Ultra-long-read sets (N50 ≥ 100 kb) produced from Oxford Nanopore sequencers have improved genome assemblies in recent years. However, despite progress in extraction protocols and library preparation methods, ultra-long sequencing remains challenging for many sample types. Here, we compare various methods and introduce the FindingNemo protocol that: (1) optimizes ultra-high-molecular-weight (UHMW) DNA extraction and library cleanup by using glass beads and hexamminecobalt(III) chloride (CoHex), (2) can deliver high ultra-long sequencing yield of >20 Gb of reads from a single MinION flow cell or >100 Gb from PromethION devices (R9.4–R10.4 pore variants), and (3) is scalable to using fewer input cells or lower DNA amounts, with extraction to sequencing possible in a single working day. By comparison, we demonstrate that this protocol surpasses previous methods by enabling precise determination of input DNA quantity and quality through cell counting, sample dilution, and homogenization techniques.

A unique feature of Oxford Nanopore Technologies sequencers, adaptive sampling, allows precise DNA molecule selection from sequencing libraries. Here, we present enhancements to our tool, readfish, enabling all features for the industrial scale PromethION sequencer, including standard and “barcode-aware” adaptive sampling. We demonstrate effective coverage enrichment and assessment of multiple human genomes for copy number and structural variation on a single PromethION flow cell.

Fewer than half of individuals with a suspected Mendelian or monogenic condition receive a precise molecular diagnosis after comprehensive clinical genetic testing. Improvements in data quality and costs have heightened interest in using long-read sequencing (LRS) to streamline clinical genomic testing, but the absence of control data sets for variant filtering and prioritization has made tertiary analysis of LRS data challenging. To address this, the 1000 Genomes Project (1KGP) Oxford Nanopore Technologies Sequencing Consortium aims to generate LRS data from at least 800 of the 1KGP samples. Our goal is to use LRS to identify a broader spectrum of variation so we may improve our understanding of normal patterns of human variation. Here, we present data from analysis of the first 100 samples, representing all 5 superpopulations and 19 subpopulations. These samples, sequenced to an average depth of coverage of 37× and sequence read N50 of 54 kbp, have high concordance with previous studies for identifying single nucleotide and indel variants outside of homopolymer regions. Using multiple structural variant (SV) callers, we identify an average of 24,543 high-confidence SVs per genome, including shared and private SVs likely to disrupt gene function as well as pathogenic expansions within disease-associated repeats that were not detected using short reads. Evaluation of methylation signatures revealed expected patterns at known imprinted loci, samples with skewed X-inactivation patterns, and novel differentially methylated regions. All raw sequencing data, processed data, and summary statistics are publicly available, providing a valuable resource for the clinical genetics community to discover pathogenic SVs.

Background: Advances in our technological capacity to interrogate brain tumour biology has led to the ever-increasing use of genomic sequencing in routine diagnostic decision making. Presently, brain tumours are routinely classified based on their epigenetic signatures, leading to a paradigm shift in diagnostic pathways. Such testing can be performed so rapidly using nanopore sequencing that results can be provided intraoperatively. This information greatly improves upon the fidelity of smear diagnosis and can help surgeons tailor their approach, balancing the risks of surgery with the likely benefit. Nevertheless, full integrated diagnosis may require subsequent additional assays to detect pathognomonic somatic mutations and structural variants, thereby delaying the time to final diagnosis. Methods: Here, we present ROBIN, a tool based upon PromethION nanopore sequencing technology that can provide both real-time, intraoperative methylome classification and next-day comprehensive molecular profiling within a single assay. ROBIN uniquely integrates three methylation classifiers ^1–3 to improve diagnostic performance in the intraoperative setting. Findings: We demonstrate classifier performance on 50 prospective intraoperative cases, achieving a diagnostic turnaround time under 2 hours and generating robust tumour classifications within minutes of sequencing. Furthermore, ROBIN can detect single nucleotide variants (SNVs), copy number variants (CNVs) and structural variants (SVs) in real-time, and is able to inform a complete integrated diagnosis within 24 hours. Classifier performance demonstrated concordance with final integrated diagnosis in 90% of prospective cases. Interpretation: Nanopore sequencing can greatly improve upon the turnaround times for standard of care diagnostic testing, including sequencing, and is furthermore able to reliably provide clinically actionable intraoperative tumour classification. Funding: The Jean-Shanks Foundation, the Pathological Society of Great Britain and Ireland, the British Neuropathological Society, and the Wellcome Trust.