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At least 19 recordsLinked to original sources
PubMed2026

VisPan: real-time visualisation of multiplex amplicon-based sequencing panels for rapid syndromic surveillance and pathogen detection.

MOTIVATION: Infectious diseases persist as a major global public health challenge. Diverse factors, including climate change, globalization, deforestation, human-animal interactions, lifestyle choices, and various biological factors, can contribute to their emergence and reemergence. Rapid detection and characterization of (re)emerging pathogens are therefore critical for effective outbreak management and for enhancing our understanding of epidemics by monitoring the transmission, spread, evolution, and genomics of pathogens. In this context, next-generation sequencing technologies (NGS), particularly long-read platforms such as Oxford Nanopore Technologies (ONT), have opened new avenues for real-time pathogen monitoring. However, the bioinformatics bottleneck remains a challenge, emphasizing the need for efficient, accessible, and user-friendly analysis tools. RESULTS: Here, we present a tool adapted from the RAMPART software that enables real-time data visualisation of multiplex PCR syndromic panels combined with Oxford Nanopore sequencing. This real-time analysis enables rapid pathogen detection, from raw data acquisition to taxonomic assignment, within minutes. The interface offers dynamic visual tracking of the sequencing run and amplicon coverage, facilitating immediate insights during diagnostic workflows. Validation experiments confirmed the system's reliability, accurately identifying all pathogens present in complex clinical or environmental samples. This tool provides an integrated, user-friendly solution for genomic pathogen surveillance in field or clinical settings.

Software↗
PubMed2024

An easy-to-use pipeline to analyze amplicon-based Next Generation Sequencing results of human mitochondrial DNA from degraded samples.

Genome and transcriptome examinations have become more common due to Next-Generation Sequencing (NGS), which significantly increases throughput and depth coverage while reducing costs and time. Mitochondrial DNA (mtDNA) is often the marker of choice in degraded samples from archaeological and forensic contexts, as its higher number of copies can improve the success of the experiment. Among other sequencing strategies, amplicon-based NGS techniques are currently being used to obtain enough data to be analyzed. There are some pipelines designed for the analysis of ancient mtDNA samples and others for the analysis of amplicon data. However, these pipelines pose a challenge for non-expert users and cannot often address both ancient and forensic DNA particularities and amplicon-based sequencing simultaneously. To overcome these challenges, a user-friendly bioinformatic tool was developed to analyze the non-coding region of human mtDNA from degraded samples recovered in archaeological and forensic contexts. The tool can be easily modified to fit the specifications of other amplicon-based NGS experiments. A comparative analysis between two tools, MarkDuplicates from Picard and dedup parameter from fastp, both designed for duplicate removal was conducted. Additionally, various thresholds of PMDtools, a specialized tool designed for extracting reads affected by post-mortem damage, were used. Finally, the depth coverage of each amplicon was correlated with its level of damage. The results obtained indicated that, for removing duplicates, dedup is a better tool since retains more non-repeated reads, that are removed by MarkDuplicates. On the other hand, a PMDS = 1 in PMDtools was the threshold that allowed better differentiation between present-day and ancient samples, in terms of damage, without losing too many reads in the process. These two bioinformatic tools were added to a pipeline designed to obtain both haplotype and haplogroup of mtDNA. Furthermore, the pipeline presented in the present study generates information about the quality and possible contamination of the sample. This pipeline is designed to automatize mtDNA analysis, however, particularly for ancient samples, some manual analyses may be required to fully validate results since the amplicons that used to be more easily recovered were the ones that had fewer reads with damage, indicating that special care must be taken for poor recovered samples.

DNA, Mitochondrial↗
PubMed2026

Evaluation of amplicon-based nanopore sequencing for foot-and-mouth disease viruses in clinical and environmental samples.

Foot-and-mouth disease (FMD) causes severe global economic loss, necessitating rapid viral characterization. Nanopore sequencing provides a simple, real-time workflow suitable for on-site outbreak response, addressing the limitations of conventional methods. In this study, we optimized a previously published amplicon-based protocol and used this method to characterize a diverse range of samples (vesicular fluid, epithelium, serum, nasal/oral swabs, and environmental samples) collected during FMD outbreaks in 2025 in the Republic of Korea. Of the 129 samples collected, we successfully recovered complete genomes from 37 samples and VP1 sequences from 85 samples. Amplifying the S-fragment in isolation and separately barcoding each pool of PCR amplicons markedly improved sequence recovery. Furthermore, sequencing success depended on viral load and sample type. Based on comparisons with real-time RT-PCR results, whole-genome sequence (WGS) recovery exceeded 77.3% at cycle threshold (Ct) values ≤25 across all clinical samples. In the Ct > 30 category, serum samples yielded the highest WGS recovery rates (44.4%). This rate was markedly higher than the success rates observed for epithelium (20.0%) and nasal swabs (9.1%), whereas oral swabs and environmental samples failed to yield any sequences (0%). However, VP1 recovery from environmental samples reached 80% at Ct ≤ 30 (8/10), providing an approach to enable non-invasive monitoring. These findings demonstrate that amplicon-based nanopore sequencing is a practical method for the rapid generation of genomic data during FMD outbreaks.IMPORTANCEAlthough rapid detection and genomic data analysis are crucial for effective foot-and-mouth disease (FMD) control, the collection of these data can be challenging for certain sample types and impacted by reduced viral loads that result from nationwide FMD vaccination. This study provides a practical solution through large-scale evaluation of an optimized amplicon-based nanopore sequencing protocol to enhance the sequencing success rates for both clinical and environmental samples. Using a modified protocol to enhance genome recovery, we demonstrated that sequence data could be retrieved from diverse sample types (even with high real-time RT-PCR cycle threshold values). We identified serum as the most suitable sample, with environmental sample sequencing allowing for non-invasive monitoring during outbreaks. These results support the use of nanopore sequencing for rapid genomic analysis, particularly in outbreak responses, such as rapid surveillance, emergency vaccine selection, and epidemiological monitoring.

Foot-and-Mouth Disease↗
PubMed2026

Wastewater-based sequencing of respiratory syncytial virus to investigate lineage dynamics and antigenic site mutations: a retrospective genomic epidemiology study.

BACKGROUND: Respiratory syncytial virus (RSV) infections pose a substantial health burden, particularly for clinically vulnerable populations such as infants and older adults. Although novel immunoprophylactic interventions show promise in providing protection, many countries may not have robust surveillance systems to monitor circulating RSV lineages and detect mutations that might reduce the effectiveness of these new interventions. We aimed to assess the diversity and temporal dynamics of circulating RSV lineages in urban populations through amplicon-based sequencing and analysis of wastewater extracts. METHODS: In this prospective observational wastewater-based genomic surveillance study, 32 raw influent 24-h composite samples were collected during the 2022-23 and 2023-24 RSV seasons from both Zurich and Geneva, Switzerland. We applied an RSV subtype-specific amplicon-based sequencing approach to obtain RSV-A and RSV-B sequences from all 64 samples. Mutations relative to reference genomes were identified at positions with read depth above 30. Relative abundances of RSV lineages were estimated from frequencies of lineage-signature mutations, present in greater than 90% of publicly available sequences of that lineage. FINDINGS: Relative abundances of RSV-B (2022-23) and RSV-A (2023-24) lineages were estimated over the two RSV seasons. During the 2022-23 season, the RSV-B B.D.E.1 lineage prevailed in both cities. In the 2023-24 season, multiple RSV-A lineages cocirculated, including A.D.1, A.D.3, A.D.5, and their sub-lineages. Identification and frequency estimation of mutations showed low-frequency, non-synonymous mutations in antigenic sites on the fusion gene of both RSV-A and RSV-B, some of which have not been reported in clinical sequences. The primary outcome was identification and relative abundance of RSV lineages in wastewater samples. INTERPRETATION: These findings show the potential of wastewater-based genomic surveillance to identify and track circulating RSV lineages and clinically relevant mutations. As novel RSV immunoprophylaxis measures are introduced in upcoming RSV seasons, wastewater-derived genomic RSV data provide a valuable baseline for understanding RSV diversity and future viral evolution under increased immunological pressure. FUNDING: This study was funded by the Swiss National Science Foundation and in part by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health. Funding for sample collection and processing was provided by the Swiss Federal Office of Public Health.

Humans↗
PubMed2025

Characterisation of a persistent SARS-CoV-2 infection lasting more than 750 days in a person living with HIV: a genomic analysis.

BACKGROUND: People who are immunocompromised can develop persistent SARS-CoV-2 infections. Several viral mutations accumulated during the course of such persistent infections have also been observed in prominent variants of concern (VOCs). Here, we characterise persistent infection and viral evolution of SARS-CoV-2 lasting more than 750 days in a person with advanced HIV-1 infection. METHODS: Between March, 2021, and July, 2022, eight clinical specimens were collected from a person living with HIV, neither receiving antiretroviral therapy nor virally suppressed, and presumed to have been initially infected with SARS-CoV-2 in mid-May, 2020. Viral RNA was extracted from each swab and an amplicon-based sequencing approach was used for genomic analysis of SARS-CoV-2. Variable sites were characterised at the consensus and subconsensus levels, and phylogenetic tools were applied to analyse viral evolution. Publicly available SARS-CoV-2 sequences from GenBank were leveraged to contextualise our sequenced samples and identify any potential evidence of transmission. FINDINGS: Genomes formed a monophyletic cluster in the B.1 lineage. 68 consensus and 67 subconsensus single nucleotide variants were observed over the course of infection. The intrahost clock rate remained similar to that of the interhost rate in contemporaneous community sequences (6·74 × 10-4 [95% credible interval 5·05 × 10-4 to 8·54 × 10-4] substitutions per site per year vs 6·11 × 10-4 [5·54 × 10-5 to 6·66 × 10-4]). Mutations grouped into two distinct subpopulations present throughout infection. 10 non-synonymous mutations in the spike protein gene were at positions in common with those defining the omicron lineage (BA.1 or BA.2), of which nine were present before November, 2021. Nine of 18 substitutions present throughout infection were rare in online databases, suggesting a lack of long transmission chains descending from this individual. INTERPRETATION: Convergent SARS-CoV-2 evolution, both in and outside the spike protein, observed in this study suggests parallels with the evolutionary process leading to emergence of the omicron VOC. The inferred absence of onward infections might indicate a loss of transmissibility during adaptation to a single host. Our results underscore the importance of appropriate treatment to cure persistent SARS-CoV-2 infections and monitoring them to understand how mutations contribute to viral adaptation. FUNDING: National Institute of General Medical Sciences of the National Institutes of Health, Centers for Disease Control and Prevention, the National Institute of Allergy and Infectious Diseases, MassCPR, and Morris Singer Foundation.

Humans↗
PubMed2025

Development and Validation of Amplicon-Based Protocol for Sequencing of Respiratory Syncytial Virus Genome.

The most prevalent cause of severe respiratory infections in children is the human respiratory syncytial virus (RSV). The advent of next-generation sequencing (NGS) has made it possible to incorporate this technology into pathogen monitoring and surveillance. Whole-genome sequencing (WGS) of RSV has now become a relatively widely used method for tracking viral evolution. Here we report an improved high-throughput RSV-WGS assay performed directly on clinical samples that is suitable for short-read sequencing platforms. A total of 100 RSV-positive samples collected between November 2022 and March 2024 fulfilled the inclusion cycle quantification criteria and were randomly included in the validation process. The WGS protocol was designed to amplify three distinct amplicons to cover the entire RSV genome. The protocol described here can be successfully replicated in several instances (approximately 95%) in samples with a relatively low viral load, typically corresponding to cycle of quantification values of 27-32. The amplicon-based protocol produced meaningful sequencing results in terms of median depth of coverage (more than 12000×) and median of mapped reads (> 1 × 106 reads). The sequences that had passed the filters showed a coverage of at least 98% across the entire genome, with cycle quantification values of 32. Based on the obtained data resulting in an easy-to-perform protocol helpful for the molecular epidemiology surveillance of RSV.

Humans↗
PubMed2025

Molecular Evolution and Zoonotic Potential of Muju Virus (Orthohantavirus puumalaense) in Craseomys regulus, Republic of Korea.

Orthohantavirus puumalaense causes hemorrhagic fever with renal syndrome in Europe, with Puumala virus (PUUV) as its primary representative. Muju virus (MUJV), harbored by Craseomys regulus, an Arvicolinae rodent species endemic to the Republic of Korea (ROK), is also a genotype of O. puumalaense. However, their genomic diversity and zoonotic potential remain largely unknown. To investigate their prevalence, 185 voles were collected from 23 regions of the ROK between 2012 and 2023. Serological assays detected anti-PUUV immunoglobulin G antibodies in five samples (3.1%), whereas reverse-transcription polymerase chain reaction confirmed MUJV RNA in identical specimens (2.7%). Amplicon-based nanopore sequencing facilitates near-complete genome recovery, enabling high-resolution comparative analysis. Phylogenetic analysis revealed distinct genetic lineages in Gangwon and Jeollabuk Provinces. Evolutionary rate estimates indicated greater sequence divergence in the S and L segments than in the M segment. A zoonotic risk assessment revealed that most MUJV variants exhibited moderate-to-high spillover potential. The molecular detection of MUJV in Cheorwon, Gangwon Province, expands its known geographic range and provides the first molecular evidence of MUJV circulation in this region. These findings highlight the need for continued surveillance and seroprevalence studies of MUJV to assess its potential for human exposure and public health relevance in the ROK.

Animals↗
PubMed2025

Allele Level Sequencing of Killer Cell Immunoglobulin-Like Receptor Genes Using Oxford Nanopore Long Read Sequencing.

The human Killer cell Immunoglobulin-like Receptor (KIR) genes, found on chromosome 19, encode for cell surface protein receptors that, through interaction with their ligand, modulate the action of Natural Killer (NK) cells and some subsets of T lymphocytes. KIR genes exhibit extensive variation through variable gene content, copy number, and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant, and infectious disease progression. KIR gene content has been used in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes. With the introduction of massively parallel clonal next generation sequencing and single molecule long read third generation sequencing, allele level determination of KIR genotypes has become feasible. We describe a method for amplicon-based long read sequencing on the Oxford Nanopore Technologies platform that provides largely unambiguous allele level typing of KIR genes. The method was validated using DNA extracted from 48 10th International Histocompatibility Workshop (IHWS) cell lines with previously published allele level KIR genotypes and 176 Western Australian samples previously tested for the presence or absence of KIR genes. Our long-read sequencing method was able to accurately determine KIR alleles with an overall concordance of 97%-99% with the published data. Importantly, phasing ambiguity caused by the inability to phase heterozygous base positions over long stretches of gene sequence was resolved in several samples. Thus, our long read PCR sequencing strategy can be used to determine KIR genotypes at allele resolution level.

Humans↗
PubMed2027

Workflow for Long-Read Amplicon Sequencing of Chikungunya Virus Using Oxford Nanopore Technology.

This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.

Chikungunya virus↗
PubMed2026

Whole-genome sequencing of adenovirus 41 directly from wastewater using nested overlapping PCR and MinION.

Human adenovirus F41 (HAdV-F41) is one of the leading causes of children's acute gastroenteritis and was recently linked to an outbreak of severe acute hepatitis of unknown etiology among children during 2021 to 2022. While most evidence is based on clinical data, wastewater-based epidemiology offers a community-level approach to monitoring circulating strains and enhancing outbreak preparedness. In this study, we developed an overlapping amplicon-based whole-genome sequencing approach to directly detect HAdV-F41 from archived wastewater samples, using nested PCR with 13 primer sets. Archived wastewater samples were collected between 2021 and 2022 from three treatment plants in Seattle, USA. The viral load ranged from 1.2 × 103 to 8.4 × 103 genome copies per liter. The Oxford Nanopore platform was used for whole-genome sequencing. Complete or partial (>84%) HAdV-F41 genomes were recovered from wastewater samples, with mean coverage depths ranging from 10³ to 10⁵. The consensus sequences showed more than 99% similarity to reference genomes in the NCBI database. The phylogenetic analysis revealed that 2 sequences clustered within lineage 2a and 11 within lineage 2b, reflecting that at least two sub-lineages were circulating in the community at that time. Our results demonstrate that the overlapping amplicon-based whole-genome sequencing approach using the Oxford Nanopore platform reliably recovers HAdV-F41 genomes from wastewater. This method offers high-resolution genomic surveillance of circulating, clinically relevant HAdV-F41, supporting wastewater-based epidemiology as a valuable tool for detecting emerging variants and strengthening the early warning system for future disease outbreaks.IMPORTANCEHuman adenovirus F41 is a primary cause of childhood gastroenteritis and has been linked to recent outbreaks of severe acute hepatitis in children, yet community-level genomic surveillance of this virus remains limited. This study shows that wastewater can be used to recover nearly complete HAdV-F41 genomes through a targeted overlapping-amplicon sequencing strategy on the Oxford Nanopore platform. By applying this method to archived wastewater samples, we detected the simultaneous circulation of multiple viral lineages in a large city. These findings extend wastewater-based epidemiology beyond SARS-CoV-2 and emphasize its importance for monitoring clinically significant enteric viruses. The method described here offers a scalable tool for tracking viral evolution in communities and enhancing early warning systems for future outbreaks.

Wastewater↗
PubMed2024

Expansion of Oropouche virus in non-endemic Brazilian regions: analysis of genomic characterisation and ecological drivers.

BACKGROUND: Oropouche virus (OROV) is an arbovirus endemic in the Amazon region that closely resembles other arboviruses in terms of human disease, leading to potential misdiagnoses. The virus ecology has mostly restricted its occurrence to the Amazon biome; however, after a large 2023-24 OROV epidemic in the Brazilian Amazon region, outbreaks are being reported across Brazil and in other countries in Latin America. Here, we investigate the OROV spread outside Amazonia. METHODS: In this genomic and epidemiological study, OROV cases from January, 2023, to July, 2024, provided by the General Coordination of Public Health Laboratories of Brazil on Aug 1, 2024, were compared by geographical location (Amazon vs non-Amazon) and municipal population size, and a linear mixed model was employed to assess the relationship between agricultural area size and cases. OROV-positive samples from central laboratories of five non-Amazonian Brazilian states were sequenced using an amplicon-based approach. Bayesian phylogeographical analysis was performed with near full-length viral genomes, incorporating individual travel histories when relevant. The estimated dates of viral introductions in each sampled location were then contextualised with public epidemiological data. FINDINGS: Epidemic data show that outside the Amazon region, OROV cases frequency was 3&#xb7;9-times higher in small municipalities than in large municipalities. The planted areas of some agricultural products, such as banana plantations, were positively correlated (r=0&#xb7;39, p<0&#xb7;0001) with OROV cases. The linear mixed model revealed that, besides banana, cassava also has larger (p<0&#xb7;05) planted areas in municipalities with OROV cases when compared with those with no cases. The phylogenetic analysis of 32 new OROV genomes reconstructed multiple exportation events of the newly identified reassortant lineage from the Amazon to other Brazilian regions between January and March, 2024. At least three of the previously described OROV phylogenetic clades circulating in the Amazon were the source of viral introductions. Molecular clock analysis estimated that viral introductions happened from 50 days to 100 days before detecting the outbreaks in each state. INTERPRETATION: Our results confirm that the novel OROV reassortant lineage spread from the Amazon to other regions in early 2024, successfully establishing local transmission. The fact that outbreaks were observed in small municipalities, instead of large urban centres, suggests that local ecological conditions that are ideal for OROV vector occurrence, such as the banana plantation environment, might be important factors driving its spread in Brazil. FUNDING: DECIT, CNPq, FAPEAM, and Inova-Fiocruz. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Brazil↗
PubMed2026

Development and evaluation of an ARTIC-based amplicon sequencing assay for whole-genome characterization of respiratory syncytial virus.

Respiratory syncytial virus (RSV), a ~15.2 kb negative-sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV-A and RSV-B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 de-identified remnant clinical specimens (102 RSV-A and 112 RSV-B) with RT-PCR Cq values <31 were included. RSV genomes were amplified using ARTIC-style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV-A (median 53,433&#xd7;; mean 51,076&#xd7;) than RSV-B (median 49,699&#xd7;; mean 46,945&#xd7;), whereas genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV-A and B.D.E.1 for RSV-B. For RSV-A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra- and inter-run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0-5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. Limits of detection were 4.4 TCID50/mL for RSV-A and 18.6 TCID50/mL for RSV-B. These results demonstrate that the ARTIC-based RSV WGS assay enables near real-time surveillance and strengthens data-driven public health responses to future outbreaks.IMPORTANCERSV, with two major subtypes, RSV-A and RSV-B, causes acute respiratory infections that can be severe in infants under 6 months and older adults. Current RSV surveillance at the GPHL relies on the Thermo Fisher TaqMan Gene Expression Capillary assay, which detects and subtypes RSV but lacks resolution for lineage classification and identification of emerging variants. To address this critical gap, GPHL developed and evaluated an amplicon-based WGS assay using 214 de-identified RSV clinical specimens. Genomes were amplified using ARTIC-style and custom-primer sets, with ARTIC primers showing superior performance. The assay demonstrated strong sequencing depth, genomic coverage, specificity, repeatability, reproducibility, and low limits of detection. RSV lineages were accurately determined based on genetic variation. These results establish that the ARTIC-based WGS assay enables near real-time genomic surveillance, supporting monitoring of circulating RSV strains and informing data-driven public health responses.

bioinformatics pipeline↗
PubMed2025

Amplicon-based analyses of single-nucleotide polymorphisms reveal the genetic structure of a forest insect baculovirus.

Amplicon-based next-generation sequencing (aNGS) is a powerful tool in diagnostics and genetic studies. We developed an aNGS approach to study the population structure of the Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), a specific pathogen of the spongy moth Lymantria dispar, a devastating lepidopteran pest in European, Asian, and American deciduous forests. Naturally occurring pathogens, such as LdMNPV, are frequently reported to cause epizootics and a rapid decline of insect pest populations. DNA samples of pooled LdMNPV-infected larvae from forest regions in Northern Bavaria (Germany) were subjected to whole genome sequencing (WGS) and aNGS optimization. Then, five marker regions were identified in the genome of LdMNPV for PCR amplification, covering 21 highly specific single-nucleotide polymorphism (SNP) positions that enabled comprehensive analysis at the intra- and intersample levels. These markers were used in aNGS analyses of 70 single larvae collected in 12 forest sites, followed by SNP-based hierarchical clustering on principal components (HCPC). This approach identified three LdMNPV population clusters consisting of homogenous (pure) and heterogeneous (mixed) LdMNPV samples. To explain the genetic variability within each sample, a model based on linear optimization was developed and validated by comparing the predictions from aNGS and WGS data. The analyses showed that LdMNPV from Bavarian forests carried genetic variants highly similar to those present in the commercial product Gypchek&#xae;, developed for biocontrol. The distribution of genetic characteristics showed some trends of geographic and temporal prevalence, which are indicative of short-distance and long-distance transmission. The aNGS approach offers a fast, cost-effective, and comprehensive insight into the natural population structure of LdMNPV.

insects↗
PubMed2026

Evaluation of one-step amplicon-based targeted enrichment for SARS-CoV-2 whole-genome sequencing using the Midnight amplicon scheme.

Genomic surveillance proved invaluable during the COVID-19 pandemic for tracking SARS-CoV-2 variants and guiding outbreak responses, underscoring the ongoing need to reduce whole-genome sequencing (WGS) costs and improve workflow efficiency to ensure accessibility in resource limited settings. Here, we evaluated a one-step reverse transcription polymerase chain reaction (RT-PCR) approach using the Midnight V2 primer scheme for targeted amplification of the SARS-CoV-2 genome, assessed its compatibility with Illumina sequencing, and compared its performance to a well-established two-step method. Initially, we determined optimal RT-PCR reaction conditions using the Midnight V2 primer panel for the one-step RT-PCR kit and scaled reaction volumes for both RT-PCR and library preparation. Clinical specimens (n&#x202f;=&#x202f;53) that had undergone routine WGS for surveillance purposes using the established two-step RT-PCR method were compared using the one-step RT-PCR assay. For samples with genome completeness greater than 70%, both methods gave comparable results with similar sequence coverage and 100% concordance for lineage assignment. Further investigation revealed a higher percentage of reads aligning to the SARS-CoV-2 genome with a greater depth of coverage using the one-step method compared to the two-step method. Finally, analysis of scaled one-step and library reaction volumes revealed significant cost savings for samples undergoing WGS. Overall, the results presented here verify the accuracy and reproducibility of one-step targeted amplification and offer an efficient and cost-effective workflow for routine SARS-CoV-2 genomic surveillance.

Humans↗
PubMed2026

A de novo algorithm for allele reconstruction from Oxford nanopore amplicon reads, with application to CYP2D6.

MOTIVATION: The Oxford Nanopore Technologies' sequencing platform offers a path towards bedside genomics, producing long reads that can completely cover a gene of interest, and detect any known or novel variant the gene contains. However, the analysis of these long reads to identify actionable genotypes remains challenging and typically requires customization depending on the target gene. RESULTS: Here, we describe a generic algorithm to accurately reconstruct allele sequences derived from long-reads of amplicon-based data. Rather than calling variants directly from these long-reads, our method takes a "sequence-first" approach, performing an unbiased reconstruction of the underlying amplicon sequences to generate high-confidence reconstructed allele sequences. This is done without user input of the target gene, allowing for any source amplicon to be reconstructed. These high-confidence reconstructed allele sequences are then compared to the genomic reference sequence of the gene to infer the specific diplotype present in the sample. This approach is agnostic towards the number of genes and alleles present and readily detects novel variants. We demonstrate our approach using three independent data sets for CYP2D6, a diverse and complex gene with over 175 known alleles of clinical significance. We show how our approach can accurately recover validated CYP2D6 diplotypes from 20 Coriell samples covering 14 distinct alleles, using different amplicons, flow cell versions, and depths. This includes inferring occurrences of allele duplication events from relative abundances of each allele, a critical factor for ascribing functional effects to a diplotype. Further, we demonstrate our approach's utility for other genomic regions, including HLA. AVAILABILITY: Custom code is available at the following GitHub repository, along with instructions for use and test data: https://github.com/scottdbrown/allele-reconstruction-long-read-amplicon-data. A snapshot of the code at the time of publication is available on Zenodo.org; doi 10.5281/zenodo.19716004. Raw .fastq sequence data for our three sequencing runs is available at the SRA under Bioproject PRJNA1357883 (https://www.ncbi.nlm.nih.gov/bioproject/1357883).

Alleles↗
PubMed2026

Molecular characterization of salivary cancers: Patterns of genomic alterations and potential for impact on therapeutic choices.

BACKGROUND: Salivary cancers are rare malignancies with diverse histologies, molecular landscape, and limited effective systemic therapy options. Recent tumour genomics research has identified driver alterations in salivary gland cancers that have led to personalized therapy approaches. The primary objective was to perform molecular characterization using next generation sequencing (NGS) panel and evaluate the potential impact of results on clinical decision-making and treatment outcomes. METHODS: Patients with locally advanced or incurable metastatic salivary cancers suitable for systemic therapy underwent NGS tumour testing with an amplicon-based DNA/RNA NGS panel. Patient demographics, baseline characteristics, treatment and treatment outcomes were retrospectively collected. RESULTS: From 2021 to 2024, 58 advanced salivary cancer patients underwent molecular characterization of their tumour. Baseline characteristics at diagnosis: male 60%, median age 67, most common histologies; adenoid cystic 27%, salivary duct 19% and mucoepidermoid 12%. PIK3CA alterations were the most common molecular finding across all subtypes 22% (13/58) and were enriched in salivary duct carcinoma 73% (8/11). Other alterations identified were: ERBB2 (4), EGFR (2), HRAS (3), NTRK3 (2), BRAF p.V600E (1), and RET (1). Immunohistochemistry identified androgen receptor positivity across salivary cancer subtypes in 8/19 and HER2 positivity in 2/20 tested. Twenty-two patients received systemic therapy prior to NGS results for incurable/metastatic disease, first line treatments included 69% chemotherapy, 18% anti-androgen, 9% lenvatinib, 4% trial. CONCLUSION: Molecular characterization of salivary cancers identified targetable alterations in 37% of patients. The identification of potential therapeutic targets offers the opportunity for expanded treatment options to benefit salivary gland cancer patients.

Metastatic salivary gland cancer↗
PubMed2026

Scalable medium-density genotyping platforms for cultivar identification, pedigree authentication, marker-assisted and genomic selection, and other applications in strawberry.

A broad spectrum of high-density genotyping approaches, including single-nucleotide polymorphism (SNP) arrays, genotyping-by-sequencing, and whole-genome reduced-representation sequencing, have been shown to perform well in strawberry (Fragaria &#xd7; ananassa), despite the inherent complexity of the octoploid genome. While these approaches are effective, their routine deployment in breeding programs can be constrained by cost, computational requirements, and workflow complexity. In parallel, many breeding programs continue to rely on locus-specific assays for marker-assisted selection, resulting in fragmented and inefficient genotyping strategies. Here, we describe medium-density amplicon-based genotyping platforms for strawberry designed to provide cost-effective, turnkey solutions that integrate markers used for marker-assisted selection with genome-wide markers suitable for genomic prediction in a single laboratory assay. These platforms were developed by targeting 1,650 or 4,811 target SNPs via amplicon sequencing, and are interoperable with existing high-density genotyping resources, including a widely used 50K SNP array, thereby facilitating data integration across platforms. We benchmarked their performance relative to the 50K SNP array across breeding-relevant applications, including identity and purity testing, pedigree authentication, marker-assisted selection, and genomic selection, and further evaluated the feasibility of genotype imputation to enhance genome-wide information content. Across analyses, the 1,650- and 4,811-amplicon platforms produced results comparable to higher-density platforms while substantially reducing genotyping cost and analytical overhead. This work demonstrates that targeted amplicon-based genotyping can support efficient, scalable, and integrated genome-informed breeding, enabling the routine application of both marker-assisted and genomic selection within strawberry breeding workflows. Open-source R workflows are provided to support streamlined analyses in breeding contexts.

Fragaria↗