De Novo Genome Assembly of Phormia regina (Diptera: Calliphoridae)

poster abstractPhormia regina (Meigen), commonly known as the black blow fly, is a dipteran that belongs to the family Calliphoridae (blow flies). Calliphorids play an important role in various research fields like ecology, medical studies, veterinary and forensic sciences. P. regina is one of the common forensically relevant insects in North America and is typically used to assist in estimating post-mortem intervals (PMI). To better understand the roles it plays in the numerous research fields, we aim to re-construct its genome using next generation sequencing technologies. We are specifically focusing on generating a reference genome by de novo assembly then use the genomic data to identify genetic markers (microsatellites, single nucleotide polymorphisms) that contribute to intra- and inter-population variation with regards to geographic location. DNA was extracted from five adult male and female flies and was sequenced using the Illumina HiSeq2000 sequencing platform. More than 250 million high quality reads were produced from each sex. These reads were used in the de novo genome assembly of the female, male and combined sexes. The assembled draft genomes produced approximately 251,115 contigs, 306,273 contigs, and 325,664 contigs respectively. The assembled genome sizes totaled to ~524 Mbp and ~508 Mbp for the female and male flies, respectively. Compared to the estimated genome sizes from a previous study of 529 Mbp for females and 517 Mbp for males, we can conclude that a majority of the genome sequence (~99%) is included in the assembly. Gene prediction and annotation of the draft genomes are currently in progress. The draft reference genomes assembled from this study will provide an important resource for analyzing genetic basis of variations between and among blow fly species, which will ultimately facilitate ongoing studies in various areas of research that utilize blow flies as study models. It will also be a source where reliable genomic data can be readily available and used in downstream analysis to increase the understanding of the genetic, molecular and cellular processes of blow flies

Comparative genomics of the sheep blow fly Lucilia cuprina

poster abstractInsects employ different adaptive strategies in response to selective pressures, such as competition for limited resources. Carrion insects provide the ideal case to study these fundamental processes of adaptive evolution due to the intense selective pressures placed on developing larvae with limited food resources, their widespread and abundant distributions, and the presence of geographically distinct populations with specialized adaptations. One adaptation is facultative ectoparasitism, where the insect strikes a healthy animal and feeds on the living flesh, providing a developmental advantage over competitor fly species, but causing significant harm to the host. Lucilia species, which hybridize in the wild and form geographically distinct subpopulations in other regions, are diverging, meaning that we can observe and quantify early biological adaptive processes that govern speciation as they are occurring over hundreds, instead of millions, of years. The draft genome of a North American male Lucilia cuprina fly (carrion breeder) was assembled using a combination of short and long read sequences. This genome is compared to an existing Australian draft genome (ectoparasite) by elucidating genomic structure in key adaptive processes (i.e. immune system evasion) via high-throughput re-sequencing of parasitic specimens, gene prediction and annotation. The carcass colonized by or animal parasitized by both species, with some geographic overlap, provides a semi-controlled environment within the larger context of the ecosystem to sample a large number of individuals with similar life history strategies, allowing for direct comparative studies to elucidate the correlation between structure and function in the genomes of carrion flies – allowing us to understand biological adaptation and speciation

Genome sequence of Phormia regina Meigen (Diptera: Calliphoridae): implications for medical, veterinary and forensic research

BACKGROUND: Blow flies (Diptera: Calliphoridae) are important medical, veterinary and forensic insects encompassing 8 % of the species diversity observed in the calyptrate insects. Few genomic resources exist to understand the diversity and evolution of this group. RESULTS: We present the hybrid (short and long reads) draft assemblies of the male and female genomes of the common North American blow fly, Phormia regina (Diptera: Calliphoridae). The 550 and 534 Mb draft assemblies contained 8312 and 9490 predicted genes in the female and male genomes, respectively; including > 93 % conserved eukaryotic genes. Putative X and Y chromosomes (21 and 14 Mb, respectively) were assembled and annotated. The P. regina genomes appear to contain few mobile genetic elements, an almost complete absence of SINEs, and most of the repetitive landscape consists of simple repetitive sequences. Candidate gene approaches were undertaken to annotate insecticide resistance, sex-determining, chemoreceptors, and antimicrobial peptides. CONCLUSIONS: This work yielded a robust, reliable reference calliphorid genome from a species located in the middle of a calliphorid phylogeny. By adding an additional blow fly genome, the ability to tease apart what might be true of general calliphorids vs. what is specific of two distinct lineages now exists. This resource will provide a strong foundation for future studies into the evolution, population structure, behavior, and physiology of all blow flies

Improved risk stratification of patients with atrial fibrillation: an integrated GARFIELD-AF tool for the prediction of mortality, stroke and bleed in patients with and without anticoagulation.

OBJECTIVES: To provide an accurate, web-based tool for stratifying patients with atrial fibrillation to facilitate decisions on the potential benefits/risks of anticoagulation, based on mortality, stroke and bleeding risks. DESIGN: The new tool was developed, using stepwise regression, for all and then applied to lower risk patients. C-statistics were compared with CHA2DS2-VASc using 30-fold cross-validation to control for overfitting. External validation was undertaken in an independent dataset, Outcome Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF). PARTICIPANTS: Data from 39 898 patients enrolled in the prospective GARFIELD-AF registry provided the basis for deriving and validating an integrated risk tool to predict stroke risk, mortality and bleeding risk. RESULTS: The discriminatory value of the GARFIELD-AF risk model was superior to CHA2DS2-VASc for patients with or without anticoagulation. C-statistics (95% CI) for all-cause mortality, ischaemic stroke/systemic embolism and haemorrhagic stroke/major bleeding (treated patients) were: 0.77 (0.76 to 0.78), 0.69 (0.67 to 0.71) and 0.66 (0.62 to 0.69), respectively, for the GARFIELD-AF risk models, and 0.66 (0.64-0.67), 0.64 (0.61-0.66) and 0.64 (0.61-0.68), respectively, for CHA2DS2-VASc (or HAS-BLED for bleeding). In very low to low risk patients (CHA2DS2-VASc 0 or 1 (men) and 1 or 2 (women)), the CHA2DS2-VASc and HAS-BLED (for bleeding) scores offered weak discriminatory value for mortality, stroke/systemic embolism and major bleeding. C-statistics for the GARFIELD-AF risk tool were 0.69 (0.64 to 0.75), 0.65 (0.56 to 0.73) and 0.60 (0.47 to 0.73) for each end point, respectively, versus 0.50 (0.45 to 0.55), 0.59 (0.50 to 0.67) and 0.55 (0.53 to 0.56) for CHA2DS2-VASc (or HAS-BLED for bleeding). Upon validation in the ORBIT-AF population, C-statistics showed that the GARFIELD-AF risk tool was effective for predicting 1-year all-cause mortality using the full and simplified model for all-cause mortality: C-statistics 0.75 (0.73 to 0.77) and 0.75 (0.73 to 0.77), respectively, and for predicting for any stroke or systemic embolism over 1 year, C-statistics 0.68 (0.62 to 0.74). CONCLUSIONS: Performance of the GARFIELD-AF risk tool was superior to CHA2DS2-VASc in predicting stroke and mortality and superior to HAS-BLED for bleeding, overall and in lower risk patients. The GARFIELD-AF tool has the potential for incorporation in routine electronic systems, and for the first time, permits simultaneous evaluation of ischaemic stroke, mortality and bleeding risks. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier for GARFIELD-AF (NCT01090362) and for ORBIT-AF (NCT01165710)

Two-year outcomes of patients with newly diagnosed atrial fibrillation: results from GARFIELD-AF.

AIMS: The relationship between outcomes and time after diagnosis for patients with non-valvular atrial fibrillation (NVAF) is poorly defined, especially beyond the first year. METHODS AND RESULTS: GARFIELD-AF is an ongoing, global observational study of adults with newly diagnosed NVAF. Two-year outcomes of 17 162 patients prospectively enrolled in GARFIELD-AF were analysed in light of baseline characteristics, risk profiles for stroke/systemic embolism (SE), and antithrombotic therapy. The mean (standard deviation) age was 69.8 (11.4) years, 43.8% were women, and the mean CHA2DS2-VASc score was 3.3 (1.6); 60.8% of patients were prescribed anticoagulant therapy with/without antiplatelet (AP) therapy, 27.4% AP monotherapy, and 11.8% no antithrombotic therapy. At 2-year follow-up, all-cause mortality, stroke/SE, and major bleeding had occurred at a rate (95% confidence interval) of 3.83 (3.62; 4.05), 1.25 (1.13; 1.38), and 0.70 (0.62; 0.81) per 100 person-years, respectively. Rates for all three major events were highest during the first 4 months. Congestive heart failure, acute coronary syndromes, sudden/unwitnessed death, malignancy, respiratory failure, and infection/sepsis accounted for 65% of all known causes of death and strokes for <10%. Anticoagulant treatment was associated with a 35% lower risk of death. CONCLUSION: The most frequent of the three major outcome measures was death, whose most common causes are not known to be significantly influenced by anticoagulation. This suggests that a more comprehensive approach to the management of NVAF may be needed to improve outcome. This could include, in addition to anticoagulation, interventions targeting modifiable, cause-specific risk factors for death. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

De novo genome assembly of the blow fly phormia regina (Diptera: Calliphoridae)

Phormia regina (Meigen), commonly known as the black blow fly is a dipteran that belongs to the family Calliphoridae. Calliphorids play an important role in various research fields including ecology, medical studies, veterinary and forensic sciences. P. regina, a non-model organism, is one of the most common forensically relevant insects in North America and is typically used to assist in estimating postmortem intervals (PMI). To better understand the roles P. regina plays in the numerous research fields, we re-constructed its genome using next generation sequencing technologies. The focus was on generating a reference genome through de novo assembly of high-throughput short read sequences. Following assembly, genetic markers were identified in the form of microsatellites and single nucleotide polymorphisms (SNPs) to aid in future population genetic surveys of P. regina. A total 530 million 100 bp paired-end reads were obtained from five pooled male and female P. regina flies using the Illumina HiSeq2000 sequencing platform. A 524 Mbp draft genome was assembled using both sexes with 11,037 predicted genes. The draft reference genome assembled from this study provides an important resource for investigating the genetic diversity that exists between and among blow fly species; and empowers the understanding of their genetic basis in terms of adaptations, population structure and evolution. The genomic tools will facilitate the analysis of genome-wide studies using modern genomic techniques to boost a refined understanding of the evolutionary processes underlying genomic evolution between blow flies and other insect species

Predictors of NOAC versus VKA use for stroke prevention in patients with newly diagnosed atrial fibrillation: Results from GARFIELD-AF

Introduction: A principal aim of the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) was to document changes in treatment practice for patients with newly diagnosed atrial fibrillation during an era when non–vitamin K antagonist oral anticoagulants (NOACs) were becoming more widely adopted. In these analyses, the key factors which determined the choice between NOACs and vitamin K antagonists (VKAs) are explored. Methods: Logistic least absolute shrinkage and selection operator regression determined predictors of NOAC and VKA use. Data were collected from 24,137 patients who were initiated on AC ± antiplatelet (AP) therapy (NOAC [51.4%] or VKA [48.6%]) between April 2013 and August 2016. Results: The most significant predictors of AC therapy were country, enrolment year, care setting at diagnosis, AF type, concomitant AP, and kidney disease. Patients enrolled in emergency care or in the outpatient setting were more likely to receive a NOAC than those enrolled in hospital (OR 1.16 [95% CI: 1.04-1.30], OR: 1.15 [95% CI: 1.05-1.25], respectively). NOAC prescribing seemed to be favored in lower-risk groups, namely, patients with paroxysmal AF, normotensive patients, and those with moderate alcohol consumption, but also the elderly and patients with acute coronary syndrome. By contrast, VKAs were preferentially used in patients with permanent AF, moderate to severe kidney disease, heart failure, vascular disease, and diabetes and with concomitant AP. Conclusion: GARFIELD-AF data highlight marked heterogeneity in stroke prevention strategies globally. Physicians are adopting an individualized approach to stroke prevention where NOACs are favored in patients with a lower stroke risk but also in the elderly and patients with acute coronary syndrome

Stroke prevention in patients from Latin American countries with non-valvular atrial fibrillation: Insights from the GARFIELD-AF registry

Background: Atrial fibrillation (AF) is an important preventable cause of stroke. Anticoagulation (AC) therapy can reduce this risk. However, prescribing patterns and outcomes in patients with non-valvular AF (NVAF) from Latin American countries are poorly described. Methods: Using data from the Global Anticoagulant Registry in the FIELD-AF (GARFIELD-AF), we examined the stroke prevention strategies and the 1-year outcomes in patients from four Latin American countries: Argentina, Brazil, Chile, and Mexico. Results: A total of 4162 patients (2010-2014) were included in this analysis. At the time of AF diagnosis, 39.9% of patients were prescribed vitamin K antagonists (VKA) ± antiplatelet (AP) therapy, 21.8% non-VKA oral anticoagulant (NOAC) ± AP, 24.1% AP only and 14.1% no antithrombotic treatment. The proportion of moderate-high risk patients receiving no AC therapy at participating centers was highest in Mexico (46.4%) and lowest in Chile (14.3%). During 1-year follow-up, the rates of all-cause mortality, stroke/SE and major bleeding were: 5.77 (95% CI) (5.06-6.56), 1.58 (1.23-2.02), and 0.99 (0.72-1.36) and per 100 person-years, respectively, which are higher than the global rates across all countries in GARFIELD-AF. Unadjusted rates of all-cause mortality were highest in Argentina, 6.95 (5.43-8.90), and lowest in Chile, 4.01 (2.92-5.52). Conclusions: GARFIELD-AF results describes the marked variation in the baseline characteristics and patterns of antithrombotic treatments in patients with NVAF in four Latin American countries. Over one-third of patients with a moderate-to-high risk of stroke received no AC therapy, highlighting the need for improved management of patients according to national guideline. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Quality of vitamin k antagonist control and 1-year outcomes in patients with atrial fibrillation: A global perspective from the GARFIELD-AF registry

Aims Vitamin K antagonists (VKAs) need to be individually dosed. International guidelines recommend a target range of international normalised ratio (INR) of 2.0-3.0 for stroke prevention in atrial fibrillation (AF). We analysed the time in this therapeutic range (TTR) of VKAtreated patients with newly diagnosed AF in the ongoing, global, observational registry GARFIELD-AF. Taking TTR as a measure of the quality of patient management, we analysed its relationship with 1-year outcomes, including stroke/systemic embolism (SE), major bleeding, and all-cause mortality. Methods and Results TTR was calculated for 9934 patients using 136,082 INR measurements during 1-year follow-up. The mean TTR was 55.0%; values were similar for different VKAs. 5851 (58.9%) patients had TTR&lt;65%; 4083 (41.1%) TTR≥65%. The proportion of patients with TTR≥65% varied from 16.7% in Asia to 49.4% in Europe. There was a 2.6-fold increase in the risk of stroke/SE, 1.5-fold increase in the risk of major bleeding, and 2.4-fold increase in the risk of all-cause mortality with TTR&lt;65% versus ≥65% after adjusting for potential confounders. The population attributable fraction, i.e. the proportion of events attributable to suboptimal anticoagulation among VKA users, was 47.7% for stroke/SE, 16.7% for major bleeding, and 45.4% for all-cause mortality. In patients with TTR&lt;65%, the risk of first stroke/SE was highest in the first 4 months and decreased thereafter (test for trend, p = 0.021). In these patients, the risk of first major bleed declined during follow-up (p = 0.005), whereas in patients with TTR≥65%, the risk increased over time (p = 0.027). Conclusion A large proportion of patients with AF had poor VKA control and these patients had higher risks of stroke/SE, major bleeding, and all-cause mortality. Our data suggest that there is room for improvement of VKA control in routine clinical practice and that this could substantially reduce adverse outcomes

Characteristics of patients with atrial fibrillation prescribed antiplatelet monotherapy compared with those on anticoagulants: Insights from the GARFIELD-AF registry

Aims Current atrial fibrillation (AF) guidelines discourage antiplatelet (AP) monotherapy as alternative to anticoagulants (ACs). Why AP only is still used is largely unknown. Methods and results Factors associated with AP monotherapy prescription were analysed in GARFIELD-AF, a registry of patients with newly diagnosed (6 weeks) AF and 1 investigator-determined stroke risk factor. We analysed 51 270 patients from 35 countries enrolled into five sequential cohorts between 2010 and 2016. Overall, 20.7% of patients received AP monotherapy, 52.1% AC monotherapy, and 14.1% AP AC. Most AP monotherapy (82.5%) and AC monotherapy (86.8%) patients were CHA2DS2-VASc 2. Compared with patients on AC monotherapy, AP monotherapy patients were frequently Chinese (vs. Caucasian, odds ratio 2.73) and more likely to have persistent AF (1.32), history of coronary artery disease (2.41) or other vascular disease (1.67), bleeding (2.11), or dementia (1.81). The odds for AP monotherapy increased with 5 years of age increments for patients 75 years (1.24) but decreased with age increments for patients 55-75 years (0.86). Antiplatelet monotherapy patients were less likely to have paroxysmal (0.67) or permanent AF (0.57), history of embolism (0.56), or alcohol use (0.90). With each cohort, AP monotherapy declined (P 0.0001), especially non-indicated use. AP AC and no antithrombotic therapy were unchanged. However, even in 2015 and 2016, about 50% of AP-Treated patients had no indication except AF (71% were CHA2DS2-VASc 2). Conclusion Prescribing AP monotherapy in newly diagnosed AF has declined, but even nowadays a substantial proportion of AP-Treated patients with AF have no indication for AP. All rights reserved