Implementation, adaptation and evaluation of statistical analysis techniques for next generation sequencing data

Deep sequencing is a new high‐throughput sequencing technology intended to lower the cost of DNA sequencing further than what was previously thought possible using standard methods. Analysis of sequencing data such as SAGE (serial analysis of gene expression) and microarray data has been a popular area of research in recent years. The increasing development of these different technologies and the variety of the data produced has stressed the need for efficient analysis techniques. Various methods for the analysis of sequencing data have been developed in recent years: both SAGE data, which is discrete; and microarray data, which is continuous. These include simple analysis techniques, hierarchical clustering techniques (both Bayesian and Frequentist) and various methods for finding differential expression between groups of samples. These methods range from simple comparison techniques to more complicated computational methods, which attempt to isolate the more subtle dissimilarities in the data. Various analysis techniques are used in this thesis for the analysis of unpublished deep sequencing data. This analysis was approached in three sections. The first was looking at clustering techniques previously developed for SAGE data, Poisson C / Poisson L algorithm and a Bayesian hierarchical clustering algorithm and evaluating and adapting these techniques for use on the deep sequencing data. The second was looking at methods to find differentially expressed tags in the dataset. These differentially expressed tags are of interest, as it is believed that finding tags which are significantly up or down regulatedacross groups of samples could potentially be useful in the treatment of certain diseases. Finally due to the lack of published data, a simulation study was constructed using various models to simulate the data and assess the techniques mentioned above on data with pre‐defined sample groupings and differentially expressed tags. The main goals of the simulation study were the validation of the analysis techniques previously discussed and estimation of false positive rates for this type of large, sparse dataset. The Bayesian algorithm yielded surprising results, producing no hierarchy, suggesting no evidence of clustering. However, promising results were obtained for the adapted Poisson C / Poisson L algorithm applied using various models to fit the data and measures of similarity. Further investigation is needed to confirm whether it is suitable for the clustering of deep sequencing data in general, especially where the situation of three or more groups of interest occurs. From the results of the differential expression analysis it can be deduced that the overdispersed log linear method for the analysis of differential expression, particularly when compared to simple test such as the 2‐sample t‐tests and the Wilcoxon signed rank test is the most reliable. This deduction is made based upon the results of the overlapping with other methods and the more reasonable number of differentially expressed tags detected, in contrast to those detected using the adapted log ratio method. However none of this can be confirmed, as no information was known about the tags in either dataset. The success of the Poisson C / Poisson L algorithm on both the Poisson and Truncated Poisson simulated datasets suggests that the method of simulation is acceptable for the assessment of clustering algorithms developed for use on sequencing data. However, evaluation of the differential expression analysis performed on the simulated data indicates that further work is needed on the method of simulation to increase its reliability. The algorithms presented can be adapted for use on any form of discrete data. From the work done here, there is there is evidence that the adapted Poisson C / Poisson L algorithm is a promising technique for the analysis of deep sequencing data

Mortality in intensive care: The impact of bacteremia and the utility of systemic inflammatory response syndrome

Background: The purpose of this study was to determine the impact of bacteremia on intensive care unit (ICU) mortality and to develop a bacteremia prediction tool using systemic inflammatory response syndrome (SIRS) criteria. Methods: Patients included those aged >18 years who had blood cultures taken in the ICU from January 1, 2011-December 31, 2013. Eligible patients were identified from microbiology records of the Glasgow Royal Infirmary, Scotland. Clinical and outcome data were gathered from ICU records. Patients with clinically significant bacteremia were matched to controls using propensity scores. SIRS criteria were gathered and used to create decision rules to predict the absence of bacteremia. The main outcome was mortality at ICU discharge. The utility of the decision tools was measured using sensitivity and specificity. Results: One hundred patients had a clinically significant positive blood culture and were matched to 100 controls. Patients with bacteremia had higher ICU mortality (odds ratio [OR], 2.35; P = .001) and longer ICU stay (OR, 17.0 vs 7.8 days; P ≤ .001). Of 1,548 blood culture episodes, 1,274 met ≥2 SIRS criteria (106 significant positive cultures and 1,168 negative cultures). There was no association between SIRS criteria and positive blood cultures (P = .11). A decision rule using 3 SIRS criteria had optimal predictive performance (sensitivity, 56%; specificity, 50%) but low accuracy. Conclusions: ICU patients with bacteremia have increased mortality and length of ICU stay. SIRS criteria cannot be used to identify patients at low risk of bacteremia

Exploratory analyses to guide inclusion, limitation of sample size and strengthening of endpoints in clinical stroke trials

Clinical trials for treatment of acute ischaemic stroke require large numbers of patients and are expensive to conduct. Treatment is typically administered within the first hours or days after stroke onset. Outcome is usually assessed by a single measure, the most common being the modified Rankin scale (mRS) at day 90. Any strategy that can reduce cost or deliver more reliable answers on safety and efficacy of the investigational treatment would be welcome for future exploratory testing of novel treatments. This thesis focused on the impact of applying different methods of design, inclusion and outcome measurement to limit sample size and strengthen analysis in clinical trials in acute stroke. Firstly, inclusion criteria were investigated to assess the impact on functional outcome. By assessing how the effect of thrombolysis changes over onset time to treatment (OTT) the relationship between OTT and age could be investigated. By looking across the entire range of OTT and assessing the interaction between the two covariates this provided complementary data to a previous VISTA analysis conducted by Mishra et al. It was found that across the full range of OTT, up to 3.5h, the treatment effect of thrombolysis in very elderly stroke patients (>80 years old) was comparable to that of their younger counterparts. The association of AF and modified Rankin Scale (mRS) at day 90 was then assessed. Multiple logistic regression analysis adjusted for age and baseline National Institutes of Health Stroke Scale (NIHSS) showed that history of AF had no independent impact on stroke outcome. Deferred selection of subjects for neurorestorative therapies from hyperacute (<6h) to 24h was then explored using a simulation approach. The sample size required to detect a ‘shift’ in mRS outcome equivalent to a 5% absolute difference in proportion achieving mRS 0-2 versus 3-6 was modelled, setting power at 80% and assuming adjustment for entry age and NIHSS. It was found that extending the time window for patient selection provides a measurement which has a stronger more predictive relationship with outcome. Trial inclusion was explored further by investigating selection for delayed treatment with thrombolysis. Prognostic scoring methods were proposed to identify a strategy for patient selection to be applied first to an existing trial dataset and then validated in the pooled RCT 4.5-6h data. ). Prognostic score limits were chosen to optimise the sample for a net treatment benefit significant at p=0.01 by Cochran Mantel Haenszel test and by ordinal logistic regression. More inclusive limits were also defined based on p=0.05 criteria. After finalising prognostic score limits, for validation they were applied by an independent statistician to the pooled RCT data for 4.5-6h. The validation analysis based on ordinal outcomes failed to deliver a population in whom treatment >4.5h was safe and effective; analysis based on net benefit (mRS 0-1) showed significance. Secondly, different strategies for endpoint selection were considered. In the past some trialists have investigated the use of earlier endpoints on single trial datasets and taken advantage of the fact that numerous outcome scales are available to measure various domains of neurological and functional recovery. The use of an earlier neurological endpoint for detecting futility in a trial was considered with validation on external RCT data. Global endpoints, investigating different aspects of functional recovery at different time-points were then considered. Simulations were undertaken to assess the relationship between sample size and power for ordinal scales and the corresponding global outcomes. Day 7 NIHSS was found to be the most sensitive individual ordinal endpoint. Dichotomised analyses supported these results. However this needed validation in a randomised trial dataset for use in exploratory stroke trials. The validation study reinforced the results from the non-randomised VISTA study. The global test combination of NIHSS90 with NIHSS7 appeared to offer incremental sensitivity to treatment effect compared to the ordinal scales alone. The combination of mRS90 with NIHSS7 did not increase the sensitivity to treatment effect when compared to NIHSS alone, but offers a broader clinical measure without loss of statistical power. Finally, alternatives to the traditional RCT were considered. Abandoning the rigour of the blinded RCT carries substantial penalty in loss of reliability and should not be undertaken lightly. If a placebo control is deemed impractical or unethical, investigators often consider comparisons against historical controls. A within-VISTA exploration of case-control matching is presented. The reliability of different matching methods and covariate combinations were assessed using a simulation approach. The results indicate that caution must be taken when using historical controls to generate a matched control group. Substantial further work matching to external data and validation to RCT data is needed. Cluster randomised trials, which randomise patients by groups, are becoming a more widely used approach. When evaluating strategies to promote the transfer of research findings into clinical practice, i.e. in "Implementation Research", an RCT is impractical and a cluster randomised trial design is of advantage. Some elements in the design and sample size calculation of cluster randomised trials were considered. Intra cluster correlation coefficients (ICCs) were estimated from linear and generalised linear mixed models using maximum likelihood estimation for common measures used in stroke research. These estimates of relevant ICCs should assist in the design and planning of cluster randomised trials. In conclusion, this research has shown that there are several areas in the design of clinical trials of acute stroke that merit further investigation. Several strategies have been highlighted that could potentially reduce sample size whilst retaining optimal levels of statistical power. However other aspects such as patient selection and the nature of the intervention under study can affect trial cost and statistical power and need to be taken under consideration

Risk factors of ischemic stroke and subsequent outcome in hemodialysis patients

Background and purpose: End stage renal disease (ESRD) requiring hemodialysis (HD) carries up to a 10-fold greater risk of stroke than normal renal function. Knowledge concerning risk factors and management strategies derived from the general population may not be applicable to those with ESRD. We studied a large ESRD population to identify risk factors and outcomes for stroke. Methods: All adult patients receiving HD for ESRD from 01/01/2007 to 31/12/2012 were extracted from the electronic patient record. Variables associated with stroke were identified by survival analysis; demographic, clinical, imaging and dialysis related variables were assessed and case-fatality determined. Follow-up was until 31/12/2013. Results: 1382 patients were identified (mean age 60.5 years, 58.5% male). The prevalence of AF was 21.2% and 59.4% were incident HD patients. 160 (11.6%) experienced a stroke during 3471 patient-years of follow-up (95% ischemic). Stroke incidence was 41.5/1000 patient-years in prevalent and 50.1/1000 patient-years in incident HD patients. Factors associated with stroke on regression analysis were prior stroke, diabetes and age at starting renal replacement therapy. AF was not significantly associated with stroke and warfarin did not affect stroke risk in warfarin treated patients. Fatality was 18.8% at 7, 26.9% at 28 and 56.3% 365 days after stroke.&lt;p&gt;&lt;/p&gt; Conclusions: Incidence of stroke is high in patients with ESRD on HD with high case-fatality. Incident HD patients had the highest stroke incidence. Many, but not all, important risk factors commonly associated with stroke in the general population were not associated with stroke in patients receiving HD

End-user perceptions of a patient-and family-centred intervention to improve nutrition intake among oncology patients: A descriptive qualitative analysis

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Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Rescue of deficits by Brwd1 copy number restoration in the Ts65Dn mouse model of Down syndrome

With an incidence of ~1 in 800 births, Down syndrome (DS) is the most com- mon chromosomal condition linked to intellectual disability worldwide. While the genetic basis of DS has been identified as a triplication of chromosome 21 (HSA21), the genes encoded from HSA 21 that directly contribute to cognitive de fi cits remain incompletely understood. Here, we found that the HSA21- encoded chromatin effector, BRWD1, was upregulated in neurons derived from iPS cells from an individual with Down syndrome and brain of trisomic mice. We showed that selective copy number restoration of Brwd1 in trisomic animals rescued de fi cits in hippocampal LTP, cognition and gene expression. We demonstrated that Brwd1 tightly binds the BAF chromatin remodeling complex, and that increased Brwd1 expression promotes BAF genomic mistargeting. Importantly, Brwd1 renormalization rescued aberrant BAF localization, along with associated changes in chromatin accessibility and gene expression. These findings establish BRWD1 as a key epigenomic mediator of normal neurodevelopment and an important contributor to DS-related phenotypes

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead