Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing

Next Generation Sequencing (NGS) technologies are gaining importance in the routine clinical diagnostic setting. It is thus desirable to simplify the workflow for high-throughput diagnostics. Fragmentation of DNA is a crucial step for preparation of template libraries and various methods are currently known. Here we evaluated the performance of nebulization, sonication and random enzymatic digestion of long-range PCR products on the results of NGS. All three methods produced high-quality sequencing libraries for the 454 platform. However, if long-range PCR products of different length were pooled equimolarly, sequence coverage drastically dropped for fragments below 3,000 bp. All three methods performed equally well with regard to overall sequence quality (PHRED) and read length. Enzymatic fragmentation showed highest consistency between three library preparations but performed slightly worse than sonication and nebulization with regard to insertions/deletions in the raw sequence reads. After filtering for homopolymer errors, enzymatic fragmentation performed best if compared to the results of classic Sanger sequencing. As the overall performance of all three methods was equal with only minor differences, a fragmentation method can be chosen solely according to lab facilities, feasibility and experimental design

Characteristics and outcomes of older patients hospitalised for COVID-19 in the first and second wave of the pandemic in The Netherlands:the COVID-OLD study

BACKGROUND: as the coronavirus disease of 2019 (COVID-19) pandemic progressed diagnostics and treatment changed. OBJECTIVE: to investigate differences in characteristics, disease presentation and outcomes of older hospitalised COVID-19 patients between the first and second pandemic wave in The Netherlands. METHODS: this was a multicentre retrospective cohort study in 16 hospitals in The Netherlands including patients aged ≥ 70 years, hospitalised for COVID-19 in Spring 2020 (first wave) and Autumn 2020 (second wave). Data included Charlson comorbidity index (CCI), disease severity and Clinical Frailty Scale (CFS). Main outcome was in-hospital mortality. RESULTS: a total of 1,376 patients in the first wave (median age 78 years, 60% male) and 946 patients in the second wave (median age 79 years, 61% male) were included. There was no relevant difference in presence of comorbidity (median CCI 2) or frailty (median CFS 4). Patients in the second wave were admitted earlier in the disease course (median 6 versus 7 symptomatic days; P < 0.001). In-hospital mortality was lower in the second wave (38.1% first wave versus 27.0% second wave; P < 0.001). Mortality risk was 40% lower in the second wave compared with the first wave (95% confidence interval: 28–51%) after adjustment for differences in patient characteristics, comorbidity, symptomatic days until admission, disease severity and frailty. CONCLUSIONS: compared with older patients hospitalised in the first COVID-19 wave, patients in the second wave had lower in-hospital mortality, independent of risk factors for mortality. The better prognosis likely reflects earlier diagnosis, the effect of improvement in treatment and is relevant for future guidelines and treatment decisions

Fatal Cardiac Arrhythmia and Long-QT Syndrome in a New Form of Congenital Generalized Lipodystrophy with Muscle Rippling (CGL4) Due to PTRF-CAVIN Mutations

We investigated eight families with a novel subtype of congenital generalized lipodystrophy (CGL4) of whom five members had died from sudden cardiac death during their teenage years. ECG studies revealed features of long-QT syndrome, bradycardia, as well as supraventricular and ventricular tachycardias. Further symptoms comprised myopathy with muscle rippling, skeletal as well as smooth-muscle hypertrophy, leading to impaired gastrointestinal motility and hypertrophic pyloric stenosis in some children. Additionally, we found impaired bone formation with osteopenia, osteoporosis, and atlanto-axial instability. Homozygosity mapping located the gene within 2 Mbp on chromosome 17. Prioritization of 74 candidate genes with GeneDistiller for high expression in muscle and adipocytes suggested PTRF-CAVIN (Polymerase I and transcript release factor/Cavin) as the most probable candidate leading to the detection of homozygous mutations (c.160delG, c.362dupT). PTRF-CAVIN is essential for caveolae biogenesis. These cholesterol-rich plasmalemmal vesicles are involved in signal-transduction and vesicular trafficking and reside primarily on adipocytes, myocytes, and osteoblasts. Absence of PTRF-CAVIN did not influence abundance of its binding partner caveolin-1 and caveolin-3. In patient fibroblasts, however, caveolin-1 failed to localize toward the cell surface and electron microscopy revealed reduction of caveolae to less than 3%. Transfection of full-length PTRF-CAVIN reestablished the presence of caveolae. The loss of caveolae was confirmed by Atomic Force Microscopy (AFM) in combination with fluorescent imaging. PTRF-CAVIN deficiency thus presents the phenotypic spectrum caused by a quintessential lack of functional caveolae

Strategy for the management of diabetic macular edema: the European Vitreo-Retinal Society macular edema study

Objective. To compare the efficacy of different therapies in the treatment of diabetic macular edema (DME). Design. Nonrandomized, multicenter clinical study. Participants. 86 retina specialists from 29 countries provided clinical information on 2,603 patients with macular edema including 870 patients with DME. Methods. Reported data included the type and number of treatment(s) performed, the pre-and posttreatment visual acuities, and other clinical findings.The results were analyzed by the French INSEE (National Institute of Statistics and Economic Studies). Main Outcome Measures. Mean change of visual acuity and mean number of treatments performed. Results.The change in visual acuity over time in response to each treatment was plotted in second order polynomial regression trend lines. Intravitreal triamcinolone monotherapy resulted in some improvement in vision. Treatmentwith threshold or subthreshold grid laser also resulted in minimal vision gain. Anti-VEGF therapy resulted in more significant visual improvement. Treatment with pars plana vitrectomy and internal limiting membrane (ILM) peeling alone resulted in an improvement in vision greater than that observed with anti-VEGF injection alone. In our DME study, treatment with vitrectomy and ILM peeling alone resulted in the better visual improvement compared to other therapies

Frailty is associated with in-hospital mortality in older hospitalised COVID-19 patients in the Netherlands:the COVID-OLD study

BACKGROUND: During the first wave of the coronavirus disease 2019 (COVID-19) pandemic, older patients had an increased risk of hospitalisation and death. Reports on the association of frailty with poor outcome have been conflicting. OBJECTIVE: The aim of the present study was to investigate the independent association between frailty and in-hospital mortality in older hospitalised COVID-19 patients in the Netherlands. METHODS: This was a multicentre retrospective cohort study in 15 hospitals in the Netherlands, including all patients aged ≥70 years, who were hospitalised with clinically confirmed COVID-19 between February and May 2020. Data were collected on demographics, co-morbidity, disease severity and Clinical Frailty Scale (CFS). Primary outcome was in-hospital mortality. RESULTS: A total of 1,376 patients were included (median age 78 years (interquartile range 74-84), 60% male). In total, 499 (38%) patients died during hospital admission. Parameters indicating presence of frailty (CFS 6-9) were associated with more co-morbidities, shorter symptom duration upon presentation (median 4 versus 7 days), lower oxygen demand and lower levels of C-reactive protein. In multivariable analyses, the CFS was independently associated with in-hospital mortality: compared with patients with CFS 1-3, patients with CFS 4-5 had a two times higher risk (odds ratio (OR) 2.0 (95% confidence interval (CI) 1.3-3.0)) and patients with CFS 6-9 had a three times higher risk of in-hospital mortality (OR 2.8 (95% CI 1.8-4.3)). CONCLUSIONS: The in-hospital mortality of older hospitalised COVID-19 patients in the Netherlands was 38%. Frailty was independently associated with higher in-hospital mortality, even though COVID-19 patients with frailty presented earlier to the hospital with less severe symptoms

Dietary Deoxynivalenol Contamination and Oral Lipopolysaccharide Challenge Alters the Cecal Microbiota of Broiler Chickens

Dietary deoxynivalenol (DON) impairs the intestinal functions and performance in broiler chickens, whereas little is known about the effect of DON on the gastrointestinal microbiota. This study evaluated the impact of graded levels of dietary DON contamination on the cecal bacterial microbiota, their predicted metabolic abilities and short-chain fatty acid (SCFA) profiles in chickens. In using a single oral lipopolysaccharide (LPS) challenge we further assessed whether an additional intestinal stressor would potentiate DON-related effects on the cecal microbiota. Eighty 1-day-old chicks were fed diets with increasing DON concentrations (0, 2.5, 5, and 10 mg DON per kg diet) for 5 weeks and were sampled after half of the chickens received an oral LPS challenge (1 mg LPS/kg bodyweight) 1 day before sampling. The bacterial composition was investigated by Illumina MiSeq sequencing of the V3–5 region of the 16S rRNA gene. DON-feeding decreased (p &lt; 0.05) the cecal species richness (Chao1) and evenness (Shannon) compared to the non-contaminated diet. The phyla Firmicutes and Proteobacteria tended to linearly increase and decrease with increasing DON-concentrations, respectively. Within the Firmicutes, DON decreased the relative abundance of Oscillospira, Clostridiaceae genus, Clostridium, and Ruminococcaceae genus 2 (p &lt; 0.05), whereas it increased Clostridiales genus 2 (p &lt; 0.05). Moreover, increasing DON levels linearly decreased a high-abundance Enterobacteriaceae genus and an Escherichia/Shigella-OTU (p &lt; 0.05). Changes in the bacterial composition and their imputed metagenomic capabilities may be explained by DON-related changes in host physiology and cecal nutrient availability. The oral LPS challenge only decreased the abundance of an unassigned Clostridiales genus 2 (p = 0.03). Increasing dietary concentrations of DON quadratically increased the cecal total SCFA and butyrate concentration (p &lt; 0.05), whereas a DON × LPS interaction indicated that LPS mainly increased cecal total SCFA, butyrate, and acetate concentrations in chickens fed the diets that were not contaminated with DON. The present findings showed that even the lowest level of dietary DON contamination had modulatory effects on chicken's cecal bacterial microbiota composition and diversity, whereas the additional oral challenge with LPS did not potentiate DON effects on the cecal bacterial composition

Effects of Citric and Lactic Acid on the Reduction of Deoxynivalenol and Its Derivatives in Feeds

Exposure to mycotoxin-contaminated feeds represents a serious health risk. This has necessitated the need for the establishment of practical methods for mycotoxin decontamination. This study investigated the effects of citric acid (CA) and lactic acid (LA) on common trichothecene mycotoxins in feeds contaminated with Fusarium mycotoxins. Contaminated feed samples were processed either with 5% CA or 5% LA solutions in a ratio of 1:1.2 (w/v) for 5, 24, or 48 h, and analyzed for multiple mycotoxin metabolites using a liquid chromatography–tandem mass spectrometric method. The analyses showed that treating the feed with CA and LA lowered the concentration of deoxynivalenol (DON), whereby 5% LA lowered the original DON concentration in the contaminated feed samples by half, irrespective of the processing time. Similar lowering effects were observed for the concentrations of 15Ac-DON, 5-hydroxyculmorin, and sambucinol. The concentration of nivalenol was only lowered by the LA treatment. In contrast, CA and LA treatments showed no or only small effects on the concentration of several mycotoxins and their derivatives, including zearalenone, fumonisins, and culmorin. In conclusion, the present results indicate that the use of 5% solutions of LA and CA might reduce the concentration of common trichothecene mycotoxins, especially DON and its derivate 15Ac-DON. However, further research is required to determine the effect on overall toxicity and to identify the underlying mechanisms

Data_Sheet_1_Dietary Deoxynivalenol Contamination and Oral Lipopolysaccharide Challenge Alters the Cecal Microbiota of Broiler Chickens.PDF

<p>Dietary deoxynivalenol (DON) impairs the intestinal functions and performance in broiler chickens, whereas little is known about the effect of DON on the gastrointestinal microbiota. This study evaluated the impact of graded levels of dietary DON contamination on the cecal bacterial microbiota, their predicted metabolic abilities and short-chain fatty acid (SCFA) profiles in chickens. In using a single oral lipopolysaccharide (LPS) challenge we further assessed whether an additional intestinal stressor would potentiate DON-related effects on the cecal microbiota. Eighty 1-day-old chicks were fed diets with increasing DON concentrations (0, 2.5, 5, and 10 mg DON per kg diet) for 5 weeks and were sampled after half of the chickens received an oral LPS challenge (1 mg LPS/kg bodyweight) 1 day before sampling. The bacterial composition was investigated by Illumina MiSeq sequencing of the V3–5 region of the 16S rRNA gene. DON-feeding decreased (p < 0.05) the cecal species richness (Chao1) and evenness (Shannon) compared to the non-contaminated diet. The phyla Firmicutes and Proteobacteria tended to linearly increase and decrease with increasing DON-concentrations, respectively. Within the Firmicutes, DON decreased the relative abundance of Oscillospira, Clostridiaceae genus, Clostridium, and Ruminococcaceae genus 2 (p < 0.05), whereas it increased Clostridiales genus 2 (p < 0.05). Moreover, increasing DON levels linearly decreased a high-abundance Enterobacteriaceae genus and an Escherichia/Shigella-OTU (p < 0.05). Changes in the bacterial composition and their imputed metagenomic capabilities may be explained by DON-related changes in host physiology and cecal nutrient availability. The oral LPS challenge only decreased the abundance of an unassigned Clostridiales genus 2 (p = 0.03). Increasing dietary concentrations of DON quadratically increased the cecal total SCFA and butyrate concentration (p < 0.05), whereas a DON × LPS interaction indicated that LPS mainly increased cecal total SCFA, butyrate, and acetate concentrations in chickens fed the diets that were not contaminated with DON. The present findings showed that even the lowest level of dietary DON contamination had modulatory effects on chicken's cecal bacterial microbiota composition and diversity, whereas the additional oral challenge with LPS did not potentiate DON effects on the cecal bacterial composition.</p

NGS results in comparison with classic Sanger sequencing.

<p>The seven out of 4,096 analyzed positions comprise four heterozygous SNPs as well as three homozygous positions that had been misinterpreted by at least one experiment. 1^st line: NGS sequence call after alignment and homopolymer filtering; 2^nd line: Σ, number of sequence fragments covering the respective position; 3^rd line: percentage of the calls for the major allele. Erroneous positions are highlighted in bold face.</p><p>*SAMtools prints the allele counts before it applies the homopolymer filter but calls the genotype afterwards. Allele frequencies and predicted genotype may hence differ.</p><p>**Please note that SAMtools only reports InDels supported by a sufficient number of reads. For the subsets in which this was not the case, we calculated the percentage of the RefSeq allele with all counts for both positions and display the mean coverage over both positions as Σ.</p

Workflow for fragmentation and NGS sequencing of long-range PCR fragments.

<p>(<b>A</b>) Graphical illustration of the entire workflow. The red arrows depict a measuring and DNA-quantification step. (<b>B</b>) Analysis of fragment lengths by PAGE before (left panel) and after (right panel) removal of small fragments <500 bp with AMPure™ columns. The red boxes depict the desired size range between 600 and 1,000 bp. Neb, nebulization; Son, sonication; Enz, enzymatic fragmentation.</p