Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alternatively, other approaches now aim to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions. These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.

Coming of age: ten years of next-generation sequencing technologies

We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ∼30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ∼3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ∼6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.

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

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About the National Institute for Health and Care Excellence - NICE.

A nanopore DNA sequencer is used for real-time genomic surveillance of the Ebola virus epidemic in the field in Guinea; the authors demonstrate that it is possible to pack a genomic surveillance laboratory in a suitcase and transport it to the field for on-site virus sequencing, generating results within 24 hours of sample collection. This paper reports the use of nanopore DNA sequencers (known as MinIONs) for real-time genomic surveillance of the Ebola virus epidemic, in the field in Guinea. The authors demonstrate that it is possible to pack a genomic surveillance laboratory in a suitcase and transport it to the field for on-site virus sequencing, generating results within 24 hours of sample collection. The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths1. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10−3 and 1.42 × 10−3 mutations per site per year. This is equivalent to 16–27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic2,3,4,5,6,7. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions8. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities9. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15–60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

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Real-time, portable genome sequencing for Ebola surveillance

Long-read technologies are overcoming early limitations in accuracy and throughput, broadening their application domains in genomics. Dedicated analysis tools that take into account the characteristics of long-read data are thus required, but the fast pace of development of such tools can be overwhelming. To assist in the design and analysis of long-read sequencing projects, we review the current landscape of available tools and present an online interactive database, long-read-tools.org, to facilitate their browsing. We further focus on the principles of error correction, base modification detection, and long-read transcriptomics analysis and highlight the challenges that remain.

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Opportunities and challenges in long-read sequencing data analysis.

Foodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 patients in our hospital. This outbreak was linked to community, national and European-wide cases. Hospital patients with Salmonella are at high risk, and require a rapid response. We initially investigated this outbreak by whole-genome sequencing using a novel rapid protocol on the Illumina MiSeq; we then integrated these data with whole-genome data from surveillance sequencing, thereby placing the outbreak in a national context. Additionally, we investigated the potential of a newly released sequencing technology, the MinION from Oxford Nanopore Technologies, in the management of a hospital outbreak of Salmonella. We demonstrate that rapid MiSeq sequencing can reduce the time to answer compared to the standard sequencing protocol with no impact on the results. We show, for the first time, that the MinION can acquire clinically relevant information in real time and within minutes of a DNA library being loaded. MinION sequencing permits confident assignment to species level within 20 min. Using a novel streaming phylogenetic placement method samples can be assigned to a serotype in 40 min and determined to be part of the outbreak in less than 2 h. Both approaches yielded reliable and actionable clinical information on the Salmonella outbreak in less than half a day. The rapid availability of such information may facilitate more informed epidemiological investigations and influence infection control practices.

/pdf/rapid-draft-sequencing-and-real-time-nanopore-sequencing-in-4yhmqmf0ip.pdf

Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella

Objectives: Multidrug-resistant Enterobacteriaceae pose a significant threat to public health. We aimed to study the impact of sewage treatment effluent on antibiotic resistance reservoirs in a river.

Methods: River sediment samples were taken from downstream and upstream of a waste water treatment plant (WWTP) in 2009 and 2011. Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae were enumerated. PCR-based techniques were used to elucidate mechanisms of resistance, with a new two-step PCR-based assay developed to investigate blaCTX-M-15 mobilization. Conjugation experiments and incompatibility replicon typing were used to investigate plasmid ecology.

Results: We report the first examples of blaCTX-M-15 in UK river sediment; the prevalence of blaCTX-M-15 was dramatically increased downstream of the WWTP. Ten novel genetic contexts for this gene were identified, carried in pathogens such as Escherichia coli ST131 as well as indigenous aquatic bacteria such as Aeromonas media. The blaCTX-M-15 ­gene was readily transferable to other Gram-negative bacteria. We also report the first finding of an imipenem-resistant E. coli in a UK river.

Conclusions: The high diversity and host range of novel genetic contexts proves that evolution of novel combinations of resistance genes is occurring at high frequency and has to date been significantly underestimated. We have identified a worrying reservoir of highly resistant enteric bacteria in the environment that poses a threat to human and animal health.

Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment

http://pure-oai.bham.ac.uk/ws/files/16481315/Hawkey_Functional_metagenomic_analysis_Veterinary_Microbiology_2014.pdf

Functional metagenomic analysis reveals rivers are a reservoir for diverse antibiotic resistance genes.

https://research.birmingham.ac.uk/portal/files/18372444/Hawkey_Multidrug_resistant_Gram_negative_bacteria_Journal_Hospital_Infection_2015_1_.pdf

Multidrug-resistant Gram-negative bacteria: a product of globalization

Staphylococcus Aureus: Staphylococcus Coagulase Negative Alpha-Haemolytic Streptococci: Beta-Haemolytic Streptococci: Pneumoccus: Enteroccus Corynebacterium Listeria And Erysipelothrix Bacillus The Genus Mycobacterium: Mycobacterium Tuberculosis: Mycobacterium Other Than Tuberculosis Moraxella and Kingella: Neisseria: Neisseria Meningitidis Haemophilus Bordetella Brucella Legionella Enterobacteriacae: E. Coli And Shigella: Salmonella: Klebsiella, Enterobacter and Serratia: Proteeae and others: Yersinia: Vibrio Actinetobacter And Other Gram and Bacteria Pseudomonas Actinobacillus, Capnocytophaga, Francisella, Cardiobacterium, Pasteurella Aeromonas/Plesiomonas.

Peter M. Hawkey

Papers

Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella

Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment

Functional metagenomic analysis reveals rivers are a reservoir for diverse antibiotic resistance genes.

Multidrug-resistant Gram-negative bacteria: a product of globalization

Principles and Practice of Clinical Bacteriology