Rapid turnaround multiplex sequencing of SARS-CoV-2: comparing tiling amplicon protocol performance

Genome sequencing is pivotal to SARS-CoV-2 surveillance, elucidating the emergence and global dissemination of acquired genetic mutations. Amplicon sequencing has proven very effective for sequencing SARS-CoV-2, but prevalent mutations disrupting primer binding sites have necessitated the revision of sequencing protocols in order to maintain performance for emerging virus lineages. We compared the performance of Oxford Nanopore Technologies (ONT) Midnight and ARTIC tiling amplicon protocols using 196 Delta lineage SARS-CoV-2 clinical specimens, and 71 mostly Omicron lineage samples with S gene target failure (SGTF), reflecting circulating lineages in the United Kingdom during December 2021. 96-plexed nanopore sequencing was used. For Delta lineage samples, ARTIC v4 recovered the greatest proportion of >=90% complete genomes (81.1%; 159/193), followed by Midnight (71.5%; 138/193) and ARTIC v3 (34.1%; 14/41). Midnight protocol however yielded higher average genome recovery (mean 98.8%) than ARTIC v4 (98.1%) and ARTIC v3 (75.4%), resulting in less ambiguous final consensus assemblies overall. Explaining these observations were ARTIC v4's superior genome recovery in low viral titre/high cycle threshold (Ct) samples and inferior performance in high titre/low Ct samples, where Midnight excelled. We evaluated Omicron sequencing performance using a revised Midnight primer mix alongside the latest ARTIC v4.1 primers, head-to-head with the existing commercially available Midnight and ARTIC v4 protocols. The revised protocols both improved considerably the recovery of Omicron genomes and exhibited similar overall performance to one another. Revised Midnight protocol recovered >=90% complete genomes for 85.9% (61/71) of Omicron samples vs. 88.7% (63/71) for ARTIC v4.1. Approximate cost per sample for Midnight (12GBP) is lower than ARTIC (16GBP) while hands-on time is considerably lower for Midnight (~7 hours) than ARTIC protocols (~9.5 hours).

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Evaluation of the microbial community structure of potable water samples from occupied and unoccupied buildings using16S rRNA amplicon sequencing

10.1101/2020.07.17.209346 ◽

2020 ◽

Author(s):

Kimothy L Smith ◽

Howard A Shuman ◽

Douglas Findeisen

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Water Samples ◽

Ad Hoc ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

The Other ◽

Water Usage ◽

16S Rrna Amplicon Sequencing ◽

Oxford Nanopore

AbstractWe conducted two studies of water samples from buildings with normal occupancy and water usage compared to water from buildings that were unoccupied with little or no water usage due to the COVID-19 shutdown. Study 1 had 52 water samples obtained ad hoc from buildings in four metropolitan locations in different states in the US and a range of building types. Study 2 had 36 water samples obtained from two buildings in one metropolitan location with matched water sample types. One of the buildings had been continuously occupied, and the other substantially vacant for approximately 3 months. All water samples were analyzed using 16S rRNA amplicon sequencing with a MinION from Oxford Nanopore Technologies. More than 127 genera of bacteria were identified, including genera with members that are known to include more than 50 putative frank and opportunistic pathogens. While specific results varied among sample locations, 16S rRNA amplicon abundance and the diversity of bacteria were higher in water samples from unoccupied buildings than normally occupied buildings as was the abundance of sequenced amplicons of genera known to include pathogenic bacterial members. In both studies Legionella amplicon abundance was relatively small compared to the abundance of the other bacteria in the samples. Indeed, when present, the relative abundance of Legionella amplicons was lower in samples from unoccupied buildings. Legionella did not predominate in any of the water samples and were found, on average, in 9.6% of samples in Study 1 and 8.3% of samples in Study 2.SynopsisComparison of microbial community composition in the plumbing of occupied and unoccupied buildings during the COVID-19 pandemic shutdown.

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Nanopore amplicon sequencing reveals molecular convergence and local adaptation of opsin genes

10.1101/2020.06.13.150334 ◽

2020 ◽

Author(s):

Katherine M. Eaton ◽

Moisés A. Bernal ◽

Nathan J.C. Backenstose ◽

Trevor J. Krabbenhoft

Keyword(s):

Local Adaptation ◽

Environmental Gradients ◽

Parallel Evolution ◽

Amplicon Sequencing ◽

Species Flock ◽

Closely Related Species ◽

Oxford Nanopore ◽

Opsin Genes ◽

Potential Case ◽

Species Specific

AbstractLocal adaptation can drive diversification of closely related species across environmental gradients and promote convergence of distantly related taxa that experience similar conditions. We examined a potential case of adaptation to novel visual environments in a species flock (Great Lakes salmonids, genus Coregonus) using a new amplicon genotyping protocol on the Oxford Nanopore Flongle. Five visual opsin genes were amplified for individuals of C. artedi, C. hoyi, C. kiyi, and C. zenithicus. Comparisons revealed species-specific differences in the coding sequence of rhodopsin (Tyr261Phe substitution), suggesting local adaptation by C. kiyi to the blue-shifted depths of Lake Superior. Parallel evolution and “toggling” at this amino acid residue has occurred several times across the fish tree of life, resulting in identical changes to the visual systems of distantly related taxa across replicated environmental gradients. Our results suggest that ecological differences and local adaptation to distinct visual environments are strong drivers of both evolutionary parallelism and diversification.

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HiMAP: robust Phylogenomics from Highly Multiplexed Amplicon sequencing

10.1101/213454 ◽

2017 ◽

Cited By ~ 2

Author(s):

Julian R. Dupuis ◽

Forest T. Bremer ◽

Angela Kauwe ◽

Michael San Jose ◽

Luc Leblanc ◽

...

Keyword(s):

High Throughput Sequencing ◽

Insect Pests ◽

Amplicon Sequencing ◽

Read Mapping ◽

Sister Relationship ◽

New Approach ◽

Hands On ◽

Locus Selection ◽

Molecular Phylogenetic ◽

Tephritid Fruit Flies

ABSTRACTHigh-throughput sequencing has fundamentally changed how molecular phylogenetic datasets are assembled, and phylogenomic datasets commonly contain 50-100-fold more loci than those generated using traditional Sanger-based approaches. Here, we demonstrate a new approach for building phylogenomic datasets using single tube, highly multiplexed amplicon sequencing, which we name HiMAP (Highly Multiplexed Amplicon-based Phylogenomics), and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and post-sequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously, requires minimal hands-on time at the bench (<1/2 day), and data analysis can be accomplished without the need for read mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including some of the most economically-important agricultural insect pests. The resulting dataset (>150,000 bp concatenated alignment) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status ofZeugodacusand the sister relationship betweenDacusandZeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting dataset provides into relationships of this diverse insect group.

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The Clinical Innovations Conference

Primary Dental Care ◽

10.1308/1355761042224666 ◽

2004 ◽

Vol os11 (4) ◽

pp. 114-114

Author(s):

Kenneth A. Eaton

Keyword(s):

United Kingdom ◽

North America ◽

Management Team ◽

Practice Management ◽

New Materials ◽

The United Kingdom ◽

Hands On

At the end of May, an impressive team of speakers from North America and the United Kingdom provided two days of lectures and hands-on sessions that updated delegates on practice management, team dentistry, fixed and removable prosthodontics, endodontics and implantology, with the emphasis on new materials and techniques.

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A high throughput deep amplicon sequencing method to show the emergence and spread of Calicophoron daubneyi in United Kingdom cattle herds

10.1101/521229 ◽

2019 ◽

Cited By ~ 2

Author(s):

Neil D. Sargison ◽

Kashif Shahzad ◽

Stella Mazeri ◽

Umer Chaudhry

Keyword(s):

United Kingdom ◽

Anthelmintic Resistance ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Animal Management ◽

The United Kingdom ◽

Sequencing Method ◽

Cattle Herds ◽

The Impact ◽

Cox 1

AbstractThe prevalence of C. daubneyi infection in the United Kingdom has increased, but despite the potential for rumen flukes to cause production loss in ruminant livestock, understanding of their emergence and spread is poor. Here we describe the development of a method to explore the multiplicity of C. daubneyi infection and patterns of the parasite’s emergence and spread, based on Illumina MiSeq deep sequencing of meta barcoded amplicons of a fragment of the mt-COX-1 locus. Our results show high levels of genetic diversity per infection and between populations of 10 to 47 of adult C. daubneyi, each from a total of 32 finished prime cattle consigned to slaughter from northern United Kingdom; with 18 unique mt-COX-1 haplotypes. This has implications for the adaptability of environmental and intermediate host stages of the parasite to changing climatic and animal management conditions, or of parasitic stages to exposure to anthelmintic drugs; potentially allowing for greater pathogenicity, or the development of anthelmintic resistance, respectively. Our results illustrate the impact of high levels of animal movements in the United Kingdom, whereby multiple common mt-COX-1 haplotypes were identified in 26 populations in the absence of geographical clustering of clades.

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Integrated genomic view of SARS-CoV-2 in India

Wellcome Open Research ◽

10.12688/wellcomeopenres.16119.1 ◽

2020 ◽

Vol 5 ◽

pp. 184 ◽

Cited By ~ 1

Author(s):

Pramod Kumar ◽

Rajesh Pandey ◽

Pooja Sharma ◽

Mahesh S. Dhar ◽

Vivekanand A. ◽

...

Keyword(s):

Host Response ◽

Genomic Data ◽

Amplicon Sequencing ◽

Climatic Conditions ◽

Molecular Surveillance ◽

Multiple Introductions ◽

Oxford Nanopore ◽

The World ◽

The Usa ◽

Local Transmission

Background: India first detected SARS-CoV-2, causal agent of COVID-19 in late January 2020, imported from Wuhan, China. From March 2020 onwards, the importation of cases from countries in the rest of the world followed by seeding of local transmission triggered further outbreaks in India. Methods: We used ARTIC protocol-based tiling amplicon sequencing of SARS-CoV-2 (n=104) from different states of India using a combination of MinION and MinIT sequencing from Oxford Nanopore Technology to understand how introduction and local transmission occurred. Results: The analyses revealed multiple introductions of SARS-CoV-2 genomes, including the A2a cluster from Europe and the USA, A3 cluster from Middle East and A4 cluster (haplotype redefined) from Southeast Asia (Indonesia, Thailand and Malaysia) and Central Asia (Kyrgyzstan). The local transmission and persistence of genomes A4, A2a and A3 was also observed in the studied locations. The most prevalent genomes with patterns of variance (confined in a cluster) remain unclassified, and are here proposed as A4-clade based on its divergence within the A cluster. Conclusions: The viral haplotypes may link their persistence to geo-climatic conditions and host response. Multipronged strategies including molecular surveillance based on real-time viral genomic data is of paramount importance for a timely management of the pandemic.

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Genomic epidemiology identifies emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States

10.1101/2021.02.06.21251159 ◽

2021 ◽

Cited By ~ 3

Author(s):

Nicole L. Washington ◽

Karthik Gangavarapu ◽

Mark Zeller ◽

Alexandre Bolze ◽

Elizabeth T. Cirulli ◽

...

Keyword(s):

United States ◽

Transmission Rate ◽

Growth Dynamics ◽

The United States ◽

Gene Target ◽

Genomic Epidemiology ◽

The United Kingdom ◽

Rapid Transmission ◽

Decisive Action ◽

The U.S

SummaryAs of January of 2021, the highly transmissible B.1.1.7 variant of SARS-CoV-2, which was first identified in the United Kingdom (U.K.), has gained a strong foothold across the world. Because of the sudden and rapid rise of B.1.1.7, we investigated the prevalence and growth dynamics of this variant in the United States (U.S.), tracking it back to its early emergence and onward local transmission. We found that the RT-qPCR testing anomaly of S gene target failure (SGTF), first observed in the U.K., was a reliable proxy for B.1.1.7 detection. We sequenced 212 B.1.1.7 SARS-CoV-2 genomes collected from testing facilities in the U.S. from December 2020 to January 2021. We found that while the fraction of B.1.1.7 among SGTF samples varied by state, detection of the variant increased at a logistic rate similar to those observed elsewhere, with a doubling rate of a little over a week and an increased transmission rate of 35-45%. By performing time-aware Bayesian phylodynamic analyses, we revealed several independent introductions of B.1.1.7 into the U.S. as early as late November 2020, with onward community transmission enabling the variant to spread to at least 30 states as of January 2021. Our study shows that the U.S. is on a similar trajectory as other countries where B.1.1.7 rapidly became the dominant SARS-CoV-2 variant, requiring immediate and decisive action to minimize COVID-19 morbidity and mortality.

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Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens

BMC Genomics ◽

10.1186/s12864-020-6557-5 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Robert Player ◽

Kathleen Verratti ◽

Andrea Staab ◽

Christopher Bradburne ◽

Sarah Grady ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Amplicon Sequencing ◽

Oxford Nanopore ◽

Pcr Assays

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A Brief Survey Of The Impact Of Restructuring Of Clinical Placement And Education Delivery For Medical Students In 5 Boroughs Partnership Nhs Foundation Trust

European Psychiatry ◽

10.1016/j.eurpsy.2016.01.1586 ◽

2016 ◽

Vol 33 (S1) ◽

pp. S437-S437

Author(s):

S. Kumar ◽

A. Baldwin

Keyword(s):

Medical Students ◽

Recruitment Rate ◽

Structured Learning ◽

Attendance Rate ◽

Survey Tool ◽

The United Kingdom ◽

Hands On ◽

Competing Interest ◽

Education Delivery ◽

The Impact

IntroductionIn the United Kingdom, a drive has been initiated to duly attract and recruit medical student into career psychiatry. However, most medical students attending university find the psychiatry lacks structure and organised model in teaching delivery.As a result, many medical students put off earlier on in their studies by psychiatry.AimWe wanted to test the above hypothesis with the introduction of structure by allocating de-centralised teaching and hands on clinical experience for medical students.MethodThe collective survey tool is devised to reflect the needs of medical students with de-centralised teaching separate but to complement university studies. This included introduction of induction package, set out syllabus, structured learning sessions. The results of the feedback from the medical students were collated and the results are discussed.DiscussionThe results indicate the success of the decentralized and borough based teaching, with improved interest in psychiatry and higher attendance rate. The feedback has been positive, and efforts of the tutors have been appreciated. Whether this will translate into higher recruitment rate number is to be seen.Disclosure of interestThe authors have not supplied their declaration of competing interest.

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Niche and local geography shape the pangenome of wastewater- and livestock-associated Enterobacteriaceae

Science Advances ◽

10.1126/sciadv.abe3868 ◽

2021 ◽

Vol 7 (15) ◽

pp. eabe3868

Author(s):

Liam P. Shaw ◽

Kevin K. Chau ◽

James Kavanagh ◽

Manal AbuOun ◽

Emma Stubberfield ◽

...

Keyword(s):

Escherichia Coli ◽

Selective Pressure ◽

Natural Populations ◽

Insertion Sequences ◽

Environmental Niche ◽

Time Points ◽

The United Kingdom ◽

Diverse Species ◽

Complete Genomes ◽

Show Evidence

Escherichia coli and other Enterobacteriaceae are diverse species with “open” pangenomes, where genes move intra- and interspecies via horizontal gene transfer. However, most analyses focus on clinical isolates. The pangenome dynamics of natural populations remain understudied, despite their suggested role as reservoirs for antimicrobial resistance (AMR) genes. Here, we analyze near-complete genomes for 827 Enterobacteriaceae (553 Escherichia and 274 non-Escherichia spp.) with 2292 circularized plasmids in total, collected from 19 locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30-km radius at three time points over a year. We find different dynamics for chromosomal and plasmid-borne genes. Plasmids have a higher burden of AMR genes and insertion sequences, and AMR-gene-carrying plasmids show evidence of being under stronger selective pressure. Environmental niche and local geography both play a role in shaping plasmid dynamics. Our results highlight the importance of local strategies for controlling the spread of AMR.

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