We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, ...Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.
Should the family’s consent be sought for brain death testing? (This case is fictitious.) In mid-2022, the high profile case of Archie Battersbee raised a number of ethical and legal questions about ...the medical care of children suspected to be brain dead, particularly in the setting of family disagreement.1 2 Ultimately in Archie’s case, formal brain death testing was not possible (because he did not meet the preconditions). Proposal: informed non-dissent One potential solution would be to adopt an ‘informed non-dissent’ approach for brain death testing in children.6 This is a model that has been developed in a related context for potentially futile or medically inappropriate interventions at the end of life in critically ill children and adults.7 In such a setting, some families are emotionally unable to agree to limitations on treatment (even if they recognise that this may be in their child’s best interest).8 An informed non-dissent approach involves informing caregivers about the patient’s clinical condition and a proposed plan for further care (for example, that cardiopulmonary resuscitation would not be provided in the event of a cardiac arrest). The House of Lords has emphasised the importance of the law and ethics of medical practice moving together.17 This provides another powerful reason why the law should be interpreted in a way which permits the use of informed non-dissent to be the routine approach adopted to consent to brain death testing in this jurisdiction. In practice, as with all important issues relating to consent, the clinical record should be completed to confirm the explanation given, the parental response and the medical decision to treat informed non-dissent as implied consent to brain death testing.
Correspondence to Dr Stephen W Turner, Child Health, Royal Aberdeen Children's Hospital, Aberdeen AB25 2ZG, UK; s.w.turner@abdn.ac.uk The benefit of an integrated child health service, where patient ...care is provided by joint working across primary and secondary care, has been recognised for almost 50 years,1 and is advocated today by many institutions including the Nuffield Trust,2 the King’s Fund3 and Royal College of Paediatrics and Child Health (RCPCH).4 Three models of integrated care in a paediatric outpatient setting have been described.5 One of these three models, the ‘cluster clinic model’, found that after 12 months of being introduced first across Aberdeen City (an urban area) and then also across Aberdeenshire (a rural area), there was reduced time between referral and being seen in clinic, and an increased proportion of referrals being resolved without the need for a clinic review.6 The data we present here were collected to answer the question ‘do the initial benefits from the cluster clinic persist after 3 years across the whole of Aberdeen City and Aberdeenshire?’ The key differences between the cluster clinic and the pre-existing ‘standard’ pathways of care are: one paediatrician–general practitioner (GP) pair is allocated to an area which includes a population of ~50 000; all referrals from a cluster (geographical area which includes a group of GP practices) were vetted by one of the clinician pair; where possible, the outpatient review takes place in a community setting within the cluster area.6 Since our earlier report,6 we have experienced a number of challenges including (1) capacity for community clinics has not recovered to pre-pandemic levels, and the majority of clinics take place in the hospital; (2) regular turnover of both paediatric and GP colleagues has meant some clusters have no allocated GP; (3) not securing recurring funds to pay for GP sessional time. No system is perfect, and the evaluation has identified some limitations to this model of care. Report of the Committee on child health services: The future of child health services: new models of care. 2016.
Other UK and international models are described on the RCPCH website https://qicentral.rcpch.ac.uk/resources/systems-of-care/integrated-care and in published literature.Box 1 Descriptions of three ...models of integrated care Model 1—Connecting Care for Children (CC4C)5 Paediatric outpatient clinic time is re-purposed; each paediatrician supports a ‘neighbourhood’—here meaning a group of GP practices—covering a population of 30 000–50 000. Showcase local, national and international4 evaluations to senior decision makers in clinical, management and financial roles, for example, Integrated Care Systems in England Use quality improvement methodology to develop and spread the model, for example, https://www.ihi.org/resources/Pages/HowtoImprove/default.aspx Quantitative outcomes include cost and time efficiencies (fewer hospital appointments, tests and treatment trials) and environmental impact (reduced travel). Use value analysis such as https://www.cc4c.imperial.nhs.uk/our-experience/blog/child-health-hub-ics-value-analysis, to show that compared with adaptations made in just primary or just secondary care, these models deliver improved quality and efficiency across the entire healthcare system rather than just in one part of it. Health systems across the world need to make best use of their precious, limited resources; paediatricians need to use their time for maximum effect; families need confidence in the health system, able to reach their trusted
To explore the feasibility and acceptability of a ‘spirometry trial’, we contacted 34 principal investigators on an ongoing asthma clinical trial ...(https://www.raaceno.co.uk/Public/Public/index.cshtml), asking ‘Would your centre be able to take part in a randomised controlled trial where patients would be randomised to treatment by spirometry plus symptoms versus symptoms only?’ There was a 100% response; 26 centres would be happy to recruit patients, but 8 centres would not recruit. (DGHC) Our cross-sectional survey gives insight into the inconsistency among clinicians of the role of spirometry in managing childhood asthma. Asthma outcomes for children and young people in the UK are poor relative to Europe,4 and there is a pressing need for the role of objective testing in asthma monitoring to be rigorously assessed.
Going beyond five bases in DNA sequencing Korlach, Jonas; Turner, Stephen W
Current opinion in structural biology,
June 2012, 2012-Jun, 2012-6-00, 20120601, Volume:
22, Issue:
3
Journal Article
Peer reviewed
► DNA contains a large variety of chemical modifications to the nucleotides. ► They are increasingly appreciated to mediate important biological functions. ► Most modifications cannot be accessed ...with traditional sequencing methods. ► Several new sequencing techniques promise to directly detect many DNA modifications.
DNA sequencing has provided a wealth of information about biological systems, but thus far has focused on the four canonical bases, and 5-methylcytosine through comparison of the genomic DNA sequence to a transformed four-base sequence obtained after treatment with bisulfite. However, numerous other chemical modifications to the nucleotides are known to control fundamental life functions, influence virulence of pathogens, and are associated with many diseases. These modifications cannot be accessed with traditional sequencing methods. In this opinion, we highlight several emerging single-molecule sequencing techniques that have the potential to directly detect many types of DNA modifications as an integral part of the sequencing protocol.
We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation ...of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.
In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that ...methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N(6)-methyladenine (6 mA) and N(4)-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5'-CTAT-3'), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5'-GAN(7)TAY-3'/3'-CTN(7)ATR-5'). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.
To inform interventions focused on safely reducing urgent paediatric short stay admissions (SSAs) for convulsions.
Routinely acquired administrative data from hospital admissions in Scotland between ...2015-2017 investigated characteristics of unscheduled SSAs (an urgent admission where admission and discharge occur on the same day) for a diagnosis of febrile and/or afebrile convulsions. Semi-structured interviews to explore perspectives of health professionals (n = 19) making referral or admission decisions about convulsions were undertaken. Interpretation of mixed methods findings was complemented by interviews with four parents with experience of unscheduled SSAs of children with convulsion.
Most SSAs for convulsions present initially at hospital emergency departments (ED). In a subset of 10,588 (11%) of all cause SSAs with linked general practice data available, 72 (37%) children with a convulsion contacted both the GP and ED pre-admission. Within 30 days of discharge, 10% (n = 141) of children admitted with afebrile convulsions had been readmitted to hospital with a further convulsion. Interview data suggest that panic and anxiety, through fear that the situation is life threatening, was a primary factor driving hospital attendance and admission. Lengthy waits to speak to appropriate professionals exacerbate parental anxiety and can trigger direct attendance at ED, whereas some children with complex needs had direct access to convulsion professionals.
SSAs for convulsions are different to SSAs for other conditions and our findings could inform new efficient convulsion-specific pre and post hospital pathways designed to improve family experiences and reduce admissions and readmissions.
Numbers of urgent short stay admissions (SSAs) of children to UK hospitals are rising rapidly. This paper reports on experiences of SSAs from the perspective of parents accessing urgent care for ...their acutely unwell child and of health professionals referring, caring for, or admitting children.
A qualitative interview study was conducted by a multi-disciplinary team with patient and public involvement (PPI) to explore contextual factors relating to SSAs and better understand pre-hospital urgent care pathways. Purposive sampling of Health Board areas in Scotland, health professionals with experience of paediatric urgent care pathways and parents with experience of a SSA for their acutely unwell child was undertaken to ensure maximal variation in characteristics such as deprivation, urban-rural and hospital structure. Interviews took place between Dec 2019 and Mar 2021 and thematic framework analysis was applied.
Twenty-one parents and forty-eight health professionals were interviewed. In the context of an urgent SSA, the themes were centred around shared outcomes of care that matter. The main outcome which was common to both parents and health professionals was the importance of preserving the child's safety. Additional shared outcomes by parents and health professionals were a desire to reduce worries and uncertainty about the illness trajectory, and provide reassurance with sufficient time, space and personnel to undertake a period of skilled observation to assess and manage the acutely unwell child. Parents wanted easy access to urgent care and, preferably, with input from paediatric-trained staff. Healthcare professionals considered that it was important to reduce the number of children admitted to hospital where safe and appropriate to do so.
The shared outcomes of care between parents and health professionals emphasises the potential merit of adopting a partnership approach in identifying, developing and testing interventions to improve the acceptability, safety, efficiency, and cost-effectiveness of urgent care pathways between home and hospital.
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