Biology subjects

Loman, N. J.

Publications and source records attributed to Loman, N. J..

13 recordsLinked to original sources

bioRxiv2026

Methodological Evaluation and Data Resource for Andes Virus Sequencing Preparedness

As part of preparedness activities supporting pathogens classified under the UK High Consequence Infectious Diseases (HCID) framework, we previously evaluated both a whole-genome tiling amplicon sequencing scheme and a pan-viral hybridisation capture approach (TWIST-CVRP) for sequencing Andes virus (ANDV). In light of the recent outbreak, we make available viral sequencing datasets generated using a historical ANDV isolate (Chile, 1997). In addition, we provide an evaluation of tiling amplicon scheme performance and present recommended primer updates informed by in silico comparison with the recently released outbreak genome. These datasets are intended to support benchmarking, validation, and optimisation of bioinformatic pipelines across the community.

genomics↗

bioRxiv2025

Reducing Supply Chain Dependencies for Viral Genomic Surveillance: Get by with a Little HELP from Commercial Enzymes already in your Lab Freezer

The COVID-19 pandemic exposed vulnerabilities in global laboratory supply chains, disrupting genomic surveillance efforts essential to epidemic response. To address this challenge, we developed ARTIC HELP (Homebrew Enzymes for Library Preparation), a practical, open-source adaptation of the widely adopted ARTIC nanopore sequencing protocol for viral genomic surveillance. We describe generic, cost-effective alternatives to all enzyme mixes used in tiling multiplex RT-PCR amplification of the virus genome, and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL), making it broadly applicable to any laboratory. Through systematic evaluation, we identified a wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases as effective alternatives when standard reagents are unavailable due to high cost or limited supply: B-family Pfu-based polymerases with a fused Sso7d DNA-binding domain, and blends combining A-family (Taq-based) and B-family (Pfu-based) polymerases. Validation on clinical samples of SARS-CoV-2 and Norovirus GII confirmed that the HELP workflow achieves genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct [≤]28), the wild-type M-MLV RT combined with selected Pfu or A+B polymerases, along with optimised HELP mixes (EP, BL, AL), achieved genome coverage of 84.0-99.6%. For Norovirus GII (Ct [≤]32), the HELP workflow using one of the Pfu polymerases achieved genome coverage of >85% for six out of eight genotypes tested. Notably, several of the other polymerases tested showed reduced performance at higher Ct values. However, they still achieved strong coverage at Ct <24, supporting their use as emergency alternatives in rapid outbreak-response sequencing when viral input is high and RNA quality is sufficient. Our approach, ARTIC HELP, provides a framework which can be implemented to address supply chain disruptions, while maintaining robust genomic sequencing capabilities. A cost analysis highlights the well-known significant global disparities in reagent pricing, driven not by protocol differences but by import fees and supply barriers. Thus, our findings highlight the need for fairer global pricing models and support for local sourcing strategies like HELP, to promote equity in genomic research and ensure preparedness for future public health challenges.

genomics↗

bioRxiv2024

PrimalScheme: open-source community resources for low-cost viral genome sequencing

Viral genome sequencing using the ARTIC protocol has been a vital tool for understanding the spread of epidemics including Ebola, Zika, Covid-19 and Mpox and has seen widespread adoption due to its low cost and high sensitivity. Here, we describe PrimalScheme, an open-source toolkit and website that allows users to easily design primer schemes for amplicon sequencing of viruses, and has generated over 67,000 primer schemes for the global community since 2017. In January 2020, PrimalScheme was used to rapidly generate a primer scheme for SARS-CoV-2, with primer pools distributed to researchers from 44 countries to help scale-up genomic surveillance efforts. Overall, these primers were used to generate an estimated 18M genome sequences and the protocols were viewed online [~]250K times. To complement PrimalScheme, we have built PrimalScheme Labs, a scheme repository which allows users to find and share primer schemes as well as establishing a set of data standards. Through improvements to the primer design process, including the use of discrete primer clouds, we have expanded the use of amplicon sequencing to include diverse virus species. We demonstrate the utility of this approach through a high diversity pan-genotype Measles virus (MeV) scheme. We also demonstrate its use on a high sensitivity, short amplicon Monkeypox virus (MPXV) scheme with over 1000 primers, showing high genome recovery on low-titre clinical samples. These developments have implications for sequencing from samples such as wastewater, for genomic surveillance of endemic pathogens and in preparing for future pandemics.

genomics↗

bioRxiv2024

FindingNemo: A Toolkit for DNA Extraction, Library Preparation and Purification for Ultra Long Nanopore Sequencing

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) optimises ultra-high molecular weight (UHMW) DNA extraction and library clean-up 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 to 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 show this protocol is superior to previous ones due to precise determination of input DNA quantity and quality by cell count, sample dilution and homogenization approaches.

genomics↗

bioRxiv2020

MAJORA: Continuous integration supporting decentralised sequencing for SARS-CoV-2 genomic surveillance

Genomic epidemiology has become an increasingly common tool for epidemic response. Recent technological advances have made it possible to sequence genomes rapidly enough to inform outbreak response, and cheaply enough to justify dense sampling of even large epidemics. With increased availability of sequencing it is possible for agile networks of sequencing facilities to collaborate on the sequencing and analysis of epidemic genomic data. In response to the ongoing SARS-CoV-2 pandemic in the United Kingdom, the COVID-19 Genomics UK (COG-UK) consortium was formed with the aim of rapidly sequencing SARS-CoV-2 genomes as part of a national-scale genomic surveillance strategy. The network consists of universities, academic institutes, regional sequencing centres and the four UK Public Health Agencies. We describe the development and deployment of Majora, an encompassing digital infrastructure to address the challenge of collecting and integrating both genomic sequencing data and sample-associated metadata produced across the COG-UK network. The system was designed and implemented pragmatically to stand up capacity rapidly in a pandemic caused by a novel virus. This approach has underpinned the success of COG-UK, which has rapidly become the leading contributor of SARS-CoV-2 genomes to international databases and has generated over 60,000 sequences to date.

bioinformatics↗

bioRxiv2020

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to {pound}10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

genomics↗

bioRxiv2019

Telomere-to-telomere assembly of a complete human X chromosome

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the [~]2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

bioinformatics↗

bioRxiv2019

Early identification of dengue virus lineage replacement in Brazil using portable genomic surveillance

Over 400 million people are estimated to be at risk of acquiring dengue virus (DENV). Despite efforts to mitigate the impact of DENV epidemics, the virus remains a public health problem in the Americas: more than one million DENV cases were reported in the continent between January and July 2019 DENV was first detected in Brazil in 1982, and Brazil has reported 88% (1,127,244 cases) of all DENV cases in the Americas during 2019 to date. Sao Paulo state in the southeast of Brazil has reported nearly half of all DENV infections in the country. Here we characterised the genetic diversity of DENV strains circulating in Sao Paulo state in 2019, at the epicentre of the ongoing DENV epidemic. Using portable nanopore sequencing we generated 20 new DENV genome sequences from viremic patients with suspected dengue infection residing in two of the most-affected municipalities, Araraquara and Sao Jose do Rio Preto. We conducted a comprehensive phylogenetic analysis with 1,630 global DENV strains to better understand the evolutionary history of the DENV lineages that currently circulate in the region. The new outbreak strains were classified as DENV2 genotype III (American/Asian genotype). Notably, phylogenetic analysis indicated that the 2019 outbreak is the result of a novel DENV lineage that was recently introduced to Brazil from the Caribbean region. Our genetic analysis further indicates that the introduction and onwards spread of the outbreak lineage (named here DENV2-III BR-4) indicates a new DENV2 lineage replacement in Brazil. Dating phylogeographic analysis suggests that DENV2-III BR-4 was introduced to Brazil in or around early 2014, possibly from the Caribbean region. Our study describes the early detection of a newly introduced and rapidly-expanding DENV2 virus lineage in Brazil.\n\nAuthor SummaryDengue is the most important mosquito-borne viral disease of humans. The disease is caused by the dengue virus (DENV) that is classified within genus Flavivirus. DENV infections are caused by 4 serotypes (DENV 1-4) that are genetically related but antigenically distinct. Dengue infection results in a variety of symptoms that range from mild fever to dengue hemorrhagic fever and/or dengue shock syndrome (DHF/DSS). Clinical outcomes are associated with different types of infection, viral serotypes, genotypes, lineages, and host genetic factors. As a re-emerging infectious disease, DENV has become a serious threat to public health in the Americas, and particularly in Brazil, where it was introduced in the 1980s and became well established due to the country-wide re-infestation of the Aedes aegypti mosquito vector species. During the first six months of 2019, 1,282,183 DENV cases were reported in the Americas, with Brazil reporting a staggering 1,127,244 (88%) of all dengue cases in the continent. To date, no information exists on the genetic composition of the DENV lineage or lineages causing the current epidemic. Here we use portable sequencing to rapidly generate virus genome data from cases occurring in two different are severely-affected municipalities in Sao Paulo state, Brazil. We find that the 2019 dengue outbreak in Brazil is caused by a newly introduced DENV serotype 2 genotype III (Asian/American) that seems to be replacing previously-circulating DENV2 lineages. We discuss the potential implications of our results regarding the current outbreak in the context of previous outbreaks in the same region.

genomics↗

bioRxiv2018

Ultra-deep, long-read nanopore sequencing of mock microbial community standards

BackgroundLong sequencing reads are information-rich: aiding de novo assembly and reference mapping, and consequently have great potential for the study of microbial communities. However, the best approaches for analysis of long-read metagenomic data are unknown. Additionally, rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition.\n\nMethodsWe sequenced two commercially-available mock communities containing ten microbial species (ZymoBIOMICS Microbial Community Standards) with Oxford Nanopore GridION and PromethION. Both communities and the ten individual species isolates were also sequenced with Illumina technology.\n\nDataWe generated 14 and 16 Gbp from GridION flowcells and 146 and 148 Gbp from PromethION flowcells for the even and log communities respectively. Read length N50 was 5.3 Kbp and 5.2 Kbp for the even and log community, respectively. Basecalls and corresponding signal data are made available (4.2 TB in total).\n\nResultsAlignment to Illumina-sequenced isolates demonstrated the expected microbial species at anticipated abundances, with the limit of detection for the lowest abundance species below 50 cells (GridION). De novo assembly of metagenomes recovered long contiguous sequences without the need for pre-processing techniques such as binning.\n\nConclusionsWe present ultra-deep, long-read nanopore datasets from a well-defined mock community. These datasets will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and comparison of current laboratory and software pipelines.

bioinformatics↗

bioRxiv2018

Nanopore native RNA sequencing of a human poly(A) transcriptome

High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes from nanopore RNA data. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology.\n\nDISCLOSURESMA holds shares in Oxford Nanopore Technologies (ONT). MA is a paid consultant to ONT. REW, WT, TG, JRT, JQ, NJL, JTS, NS, AB, MA, HEO, MJ, and ML received reimbursement for travel, accommodation and conference fees to speak at events organised by ONT. NL has received an honorarium to speak at an ONT company meeting. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore. JTS, ML and MA received research funding from ONT.

genomics↗

bioRxiv2018

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluated our multiplexed amplicon approach - PrimalSeq - to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We developed an experimental protocol and computational tool (iVar) for using PrimalSeq to measure virus diversity using Illumina and compared the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.

evolutionary biology↗

bioRxiv2017

Nanopore sequencing and assembly of a human genome with ultra-long reads

Nanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation. However until recently nanopore sequencing has been mainly applied to small genomes, due to the limited output attainable. We present nanopore sequencing and assembly of the GM12878 Utah/Ceph human reference genome generated using the Oxford Nanopore MinION and R9.4 version chemistry. We generated 91.2 Gb of sequence data ([~]30x theoretical coverage) from 39 flowcells. De novo assembly yielded a highly complete and contiguous assembly (NG50 [~]3Mb). We observed considerable variability in homopolymeric tract resolution between different basecallers. The data permitted sensitive detection of both large structural variants and epigenetic modifications. Further we developed a new approach exploiting the long-read capability of this system and found that adding an additional 5x-coverage of ultra-long reads (read N50 of 99.7kb) more than doubled the assembly contiguity. Modelling the repeat structure of the human genome predicts extraordinarily contiguous assemblies may be possible using nanopore reads alone. Portable de novo sequencing of human genomes may be important for rapid point-of-care diagnosis of rare genetic diseases and cancer, and monitoring of cancer progression. The complete dataset including raw signal is available as an Amazon Web Services Open Dataset at: https://github.com/nanopore-wgs-consortium/NA12878.

genomics↗

bioRxiv2017

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas.

genomics↗