Reflections 1998

The 1998 issue of Reflections is edited by Matthew Miller with Jennifer Carlile serving as faculty adviser. Award winners of the student poetry contest include: Emily Johnson, Matt Norman, and Jenny Rogers. Award winners of the student art contest include: Joelle Chung, Jodi Baughn, Efvem Teki, and Takiya Patrick. Jodi Baughn is the winner of the photography contest.https://digitalcommons.gardner-webb.edu/reflections/1023/thumbnail.jp

APPLICATION OF NANOPORE DATA TO THE PIMMS (Pragmatic Insertional Mutation Mapping System) SEQ PIPELINE.

Significant developments in next generation sequencing technologies have over the past 15 years allowed novel and exciting opportunities to further our understanding of genomic science. Transposons are mobile regions of DNA that occur frequently throughout the genome, the insertion of these transposable elements play an essential role in both gene regulation and evolution. Locating the positions of transposon insertion into a genome can help our understanding of the role they play in such areas as gene expression and structural variation. Random transposon mediated mutagenesis has proven to be a useful tool in the identification of essential and conditionally essential genes within various bacterial species. Various methods have been generated to identify the regions within a given genome where transposon insertion has occurred. Some of these methods involve complex experimental design in the laboratory, and a high degree of competency in bioinformatics post sequencing. PIMMS (Pragmatic Insertion Mutation Mapping System) Seq was introduced in 2016 to speed up and simplify the bioinformatic pipeline when mapping transposon insertion sites using random mutagenesis sequencing data. The bacterial samples in question undergoing random mutagenesis, followed by inverse PCR, library preparation and sequencing (using Illumina technology). The use of Nanopore sequencing technologies has steadily increased since its introduction in 2014. Although the data quality is not yet as accurate as Illumina, Nanopore sequencing does offer some unique advantages, including the ability to sequence native DNA, generate long sequencing reads, and facilitate real time data analysis. Oxford Nanopore Technologies (ONT), the company that developed nanopore sequencing also offer some unique sample enrichment options such as Cas-9 targeted sequencing which, for PIMMS could potentially be used as an alternative to inverse PCR, targeting sequences that contain known transposon motifs that flank a genomic region of interest. Nanopore sequencing data was generated using S. agalactiae insertion mutant libraries, and uploaded to the PIMMS Seq bioinformatic pipeline to generate a database of transposon insertion sites. When comparing PIMMS output data generated using Nanopore sequencing to the established Illumina short read sequencing method, although the genomic distribution of insertions where consistent, differences where observed with Nanopore sequencing identifying 405 more unique insertion events, reducing the list of genes that were considered to be essential using short read sequencing. However, any advantages of this long read sequencing method must be off set against Nanopore’s sequencing base calling accuracy, which was significantly lower than that generated using Illumina technologies. The Cas-9 Targeted library preparation from ONT has been proven to enrich for transposon sequences that flank genomic regions of interest, with on average 68.1% of all reads mapping to the insert sequence. This establishes its potential as an attractive alternative to amplification based methods. This method may significantly reduce the total amount of sequencing required to run an experiment, which in turn will reduce time and costs. Any potential wet lab sample loses are minimised due to a far smaller amount of sample processing steps. Moving forward, nanopore long read sequencing is a technology that offers some unique advantages over short read technologies, and its application to the PIMMS pipeline has been demonstrated to work well. The added option of Cas- 9 Targeted nanopore sequencing, saves not only lab time and costs, but more importantly removes any potential amplification bias

APPLICATION OF NANOPORE DATA TO THE PIMMS (Pragmatic Insertional Mutation Mapping System) SEQ PIPELINE.

Significant developments in next generation sequencing technologies have over the past 15 years allowed novel and exciting opportunities to further our understanding of genomic science. Transposons are mobile regions of DNA that occur frequently throughout the genome, the insertion of these transposable elements play an essential role in both gene regulation and evolution. Locating the positions of transposon insertion into a genome can help our understanding of the role they play in such areas as gene expression and structural variation. Random transposon mediated mutagenesis has proven to be a useful tool in the identification of essential and conditionally essential genes within various bacterial species. Various methods have been generated to identify the regions within a given genome where transposon insertion has occurred. Some of these methods involve complex experimental design in the laboratory, and a high degree of competency in bioinformatics post sequencing. PIMMS (Pragmatic Insertion Mutation Mapping System) Seq was introduced in 2016 to speed up and simplify the bioinformatic pipeline when mapping transposon insertion sites using random mutagenesis sequencing data. The bacterial samples in question undergoing random mutagenesis, followed by inverse PCR, library preparation and sequencing (using Illumina technology). The use of Nanopore sequencing technologies has steadily increased since its introduction in 2014. Although the data quality is not yet as accurate as Illumina, Nanopore sequencing does offer some unique advantages, including the ability to sequence native DNA, generate long sequencing reads, and facilitate real time data analysis. Oxford Nanopore Technologies (ONT), the company that developed nanopore sequencing also offer some unique sample enrichment options such as Cas-9 targeted sequencing which, for PIMMS could potentially be used as an alternative to inverse PCR, targeting sequences that contain known transposon motifs that flank a genomic region of interest. Nanopore sequencing data was generated using S. agalactiae insertion mutant libraries, and uploaded to the PIMMS Seq bioinformatic pipeline to generate a database of transposon insertion sites. When comparing PIMMS output data generated using Nanopore sequencing to the established Illumina short read sequencing method, although the genomic distribution of insertions where consistent, differences where observed with Nanopore sequencing identifying 405 more unique insertion events, reducing the list of genes that were considered to be essential using short read sequencing. However, any advantages of this long read sequencing method must be off set against Nanopore’s sequencing base calling accuracy, which was significantly lower than that generated using Illumina technologies. The Cas-9 Targeted library preparation from ONT has been proven to enrich for transposon sequences that flank genomic regions of interest, with on average 68.1% of all reads mapping to the insert sequence. This establishes its potential as an attractive alternative to amplification based methods. This method may significantly reduce the total amount of sequencing required to run an experiment, which in turn will reduce time and costs. Any potential wet lab sample loses are minimised due to a far smaller amount of sample processing steps. Moving forward, nanopore long read sequencing is a technology that offers some unique advantages over short read technologies, and its application to the PIMMS pipeline has been demonstrated to work well. The added option of Cas- 9 Targeted nanopore sequencing, saves not only lab time and costs, but more importantly removes any potential amplification bias

Report of the third Asian Prostate Cancer study meeting.

The Asian Prostate Cancer (A-CaP) study is an Asia-wide initiative that was launched in December 2015 in Tokyo, Japan, with the objective of surveying information about patients who have received a histopathological diagnosis of prostate cancer (PCa) and are undergoing treatment and clarifying distribution of staging, the actual status of treatment choices, and treatment outcomes. The study aims to clarify the clinical situation for PCa in Asia and use the outcomes for the purposes of international comparison. Following the first meeting in Tokyo in December 2015, the second A-CaP meeting was held in Seoul, Korea, in September 2016. This, the third A-CaP meeting, was held on October 14, 2017, in Chiang Mai, Thailand, with the participation of members and collaborators from 12 countries and regions. In the meeting, participating countries and regions presented the current status of data collection, and the A-CaP office presented a preliminary analysis of the registered cases received from each country and region. Participants discussed ongoing challenges relating to data input and collection, institutional, and legislative issues that may present barriers to data sharing, and the outlook for further patient registrations through to the end of the registration period in December 2018. In addition to A-CaP-specific discussions, a series of special lectures were also delivered on the situation for health insurance in the United States, the correlation between insurance coverage and PCa outcomes, and the outlook for robotic surgery in the Asia-Pacific region. Members also confirmed the principles of authorship in collaborative studies, with a view to publishing original articles based on A-CaP data in the future

Retrospective screening of routine respiratory samples revealed undetected community transmission and missed intervention opportunities for SARS-CoV-2 in the United Kingdom.

In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on individuals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected individual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV-2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea - also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger sample of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.Whole genome sequencing of SARS-CoV-2 was funded by COG-UK; COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research and Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute

A framework for real-time monitoring, analysis and adaptive sampling of viral amplicon nanopore sequencing

The ongoing SARS-CoV-2 pandemic demonstrates the utility of real-time sequence analysis in monitoring and surveillance of pathogens. However, cost-effective sequencing requires that samples be PCR amplified and multiplexed via barcoding onto a single flow cell, resulting in challenges with maximising and balancing coverage for each sample. To address this, we developed a real-time analysis pipeline to maximise flow cell performance and optimise sequencing time and costs for any amplicon based sequencing. We extended our nanopore analysis platform MinoTour to incorporate ARTIC network bioinformatics analysis pipelines. MinoTour predicts which samples will reach sufficient coverage for downstream analysis and runs the ARTIC networks Medaka pipeline once sufficient coverage has been reached. We show that stopping a viral sequencing run earlier, at the point that sufficient data has become available, has no negative effect on subsequent down-stream analysis. A separate tool, SwordFish, is used to automate adaptive sampling on Nanopore sequencers during the sequencing run. This enables normalisation of coverage both within (amplicons) and between samples (barcodes) on barcoded sequencing runs. We show that this process enriches under-represented samples and amplicons in a library as well as reducing the time taken to obtain complete genomes without affecting the consensus sequence

Paternal low protein diet perturbs inter-generational metabolic homeostasis in a tissue-specific manner in mice

The underlying mechanisms driving paternally-programmed metabolic disease in offspring remain poorly defined. We fed male C57BL/6 mice either a control normal protein diet (NPD; 18% protein) or an isocaloric low protein diet (LPD; 9% protein) for a minimum of 8 weeks. Using artificial insemination, in combination with vasectomised male mating, we generated offspring using either NPD or LPD sperm but in the presence of NPD or LPD seminal plasma. Offspring from either LPD sperm or seminal fluid display elevated body weight and tissue dyslipidaemia from just 3 weeks of age. These changes become more pronounced in adulthood, occurring in conjunction with altered hepatic metabolic and inflammatory pathway gene expression. Second generation offspring also display differential tissue lipid abundance, with profiles similar to those of first generation adults. These findings demonstrate that offspring metabolic homeostasis is perturbed in response to a suboptimal paternal diet with the effects still evident within a second generation

Seven features of safety in maternity units: a framework based on multisite ethnography and stakeholder consultation

Background: Reducing avoidable harm in maternity services is a priority globally. As well as learning from mistakes, it is important to produce rigorous descriptions of ‘what good looks like’. Objective: We aimed to characterise features of safety in maternity units and to generate a plain language framework that could be used to guide learning and improvement. Methods: We conducted a multisite ethnography involving 401 hours of non-participant observations 33 semistructured interviews with staff across six maternity units, and a stakeholder consultation involving 65 semistructured telephone interviews and one focus group. Results: We identified seven features of safety in maternity units and summarised them into a framework, named For Us (For Unit Safety). The features include: (1) commitment to safety and improvement at all levels, with everyone involved; (2) technical competence, supported by formal training and informal learning; (3) teamwork, cooperation and positive working relationships; (4) constant reinforcing of safe, ethical and respectful behaviours; (5) multiple problem-sensing systems, used as basis of action; (6) systems and processes designed for safety, and regularly reviewed and optimised; (7) effective coordination and ability to mobilise quickly. These features appear to have a synergistic character, such that each feature is necessary but not sufficient on its own: the features operate in concert through multiple forms of feedback and amplification. Conclusions: This large qualitative study has enabled the generation of a new plain language framework—For Us—that identifies the behaviours and practices that appear to be features of safe care in hospital-based maternity units

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant

SARS-CoV-2 infections were rising during early summer 2021 in many countries associated with the Delta variant. We assessed RT-PCR swab-positivity in the REal-time Assessment of Community Transmission-1 (REACT-1) study in England. We observed sustained exponential growth with average doubling time (June-July 2021) of 25 days driven by complete replacement of Alpha variant by Delta, and by high prevalence at younger less-vaccinated ages. Unvaccinated people were three times more likely than double-vaccinated people to test positive. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination