Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome

Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio- generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism’s biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes. IMPORTANCE Studying whole-genome sequences has become an important aspect of biological research. The advent of nextgeneration sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBiogenerated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome sanalyses to facilitate functional studies into an organism’s biology

Population Genomic Analysis Reveals Differential Evolutionary Histories and Patterns of Diversity across Subgenomes and Subpopulations of Brassica napus L.

The allotetraploid species Brassica napus L. is a global crop of major economic importance, providing canola oil (seed) and vegetables for human consumption and fodder and meal for livestock feed. Characterizing the genetic diversity present in the extant germplasm pool of B. napus is fundamental to better conserve, manage and utilize the genetic resources of this species. We used sequence-based genotyping to identify and genotype 30,881 SNPs in a diversity panel of 782 B. napus accessions, representing samples of winter and spring growth habits originating from 33 countries across Europe, Asia and America. We detected strong population structure broadly concordant with growth habit and geography, and identified three major genetic groups: spring (SP), winter Europe (WE), and winter Asia (WA). Subpopulation-specific polymorphism patterns suggest enriched genetic diversity within the WA group and a smaller effective breeding population for the SP group compared to WE. Interestingly, the two subgenomes of B. napus appear to have different geographic origins, with phylogenetic analysis placing WE and WA as basal clades for the other subpopulations in the C and A subgenomes, respectively. Finally, we identified 16 genomic regions where the patterns of diversity differed markedly from the genome-wide average, several of which are suggestive of genomic inversions. The results obtained in this study constitute a valuable resource for worldwide breeding efforts and the genetic dissection and prediction of complex B. napus traits

Validation of the SCID-hu Thy/Liv mouse model with four classes of licensed antiretrovirals.

BackgroundThe SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed antiretrovirals.Methodology/principal findingsEndpoint analyses for quantification of Thy/Liv implant viral load included ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug-resistance substitutions.ConclusionGiven the fidelity of such comparisons, we conclude that this highly reproducible mouse model is likely to predict clinical antiviral efficacy in humans

Acquiescence and Extremity in Cross-National Surveys: Domain Dependence and Country-Level Correlates

Likert-type rating scales are susceptible to response styles, such as acquiescence and extremity scoring. Although it is widely acknowledged that response styles can seriously invalidate findings of cross-cultural research, their theoretical underpinnings are hardly explored. The current study analyzed domain-dependency and country differences in acquiescence and extremity scoring in a large dataset of the International Social Survey Program. The hypothesis that response styles are more likely in domains with a high personal relevance compared to domains with a low personal relevance was tentatively confirmed. Correlations with various cultural, psychological, and economic variables were investigated. We found that acquiescence was negatively related to affluence, individualism, and well-being, while extremity was only negatively related to well-being. Positive associations were found between uncertainty avoidance and both acquiescence and extremity

Sarcoidosis-related small fiber neuropathy: Focus on fatigue, pain, restless legs syndrome, and cognitive function

BACKGROUND: Of all sarcoidosis patients 60-90% suffer from fatigue, 40-80% experience small fiber neuropathy, 35% suffer from cognitive impairment and 16-52% experience restless legs syndrome (RLS). The prevalence, severity and association between fatigue, pain, RLS and cognitive impairment in patients with sarcoidosis and small fiber neuropathy (SFN) is unknown and will be investigated in this study. METHODS: Healthy controls, patients with sarcoidosis with SFN and patients with sarcoidosis without SFN were compared. They completed multiple questionnaires (fatigue assessment scale (FAS), RLS rating scale, visual analogue scale (VAS) score for pain and cognitive failure questionnaire (CFQ)), to estimate the prevalence, severity, and correlation of fatigue, RLS, pain, SFN and cognitive impairment. RESULTS: Twenty healthy volunteers, 49 patients with sarcoidosis without SFN and 48 patients with sarcoidosis with SFN were included. Fatigue was the most prevalent symptom in patients with sarcoidosis with SFN (97%), followed by pain (85%), RLS (67%) and cognitive impairment (46%). Moreover, the severity of fatigue, pain, RLS and cognitive impairment was higher in patients with sarcoidosis with SFN compared with patients with sarcoidosis without SFN (resp. p=0.0006, p=0.003, p=0.02 and p=0.009). Finally, the FAS, RLS, VASmean, VASmax and CFQ showed a strong correlation with each other (R> 0.5, p<0.05). CONCLUSIONS: Sarcoidosis patients with SFN showed an increased prevalence and higher severity of fatigue, pain, RLS and cognitive impairment compared to patients with sarcoidosis without SFN. Moreover, moderate to strong correlations were found between these symptoms

Thermal threshold testing: call for a balance between the number of measurements and abnormalities in the diagnosis of sarcoidosis-associated small fiber neuropathy

INTRODUCTION: Several recent studies of diagnosing small fiber neuropathy (SFN) have shown a lack of uniformity in thermal threshold testing (TTT) or quantitative sensory testing (QST) which makes it a challenge to compare the data. It is known that the chance of finding an abnormality increases with increasing number of measurements. OBJECTIVES: With this study, we first wanted to investigate whether TTT could benefit from a new approach focusing on the balance between the number of measurements, depending on the selection of parameters and measuring sites, and on number of abnormalities (NOAs). Second, we wanted to address the role of the method of levels (MLe) in possible desensitization during TTT measurements. METHODS: One hundred seventeen participants were included (48 patients with sarcoidosis with probable SFN, 49 without SFN, and 20 healthy controls). Thermal threshold testing measurements and Small Fiber Neuropathy Screening List (SFNSL) questionnaire were used to assess SFN. RESULTS: A combination of measuring all thermal threshold parameters at both feet except for MLe showed the best diagnostic performance. Increasing TTT NOAs correlates with the severity of SFN. Adding the SFNSL questionnaire further improves diagnostic performance. DISCUSSION: Looking at TTT NOAs in all TTT parameters except for MLe at both feet should be considered as a new approach to improve the consistency and balance between the selection of TTT parameters, measuring sites, and definition of "abnormal QST." Moreover, the SFNSL questionnaire is a valuable tool to quantify SFN symptoms and could improve SFN diagnosis

Fully Automatic, Semiautomatic, and Manual Corneal Nerve Fiber Analysis in Patients With Sarcoidosis

PURPOSE: No guidelines are available on the preferred method for analyzing corneal confocal microscopy (CCM) data. Manual, semiautomatic, and automatic analyzes are all currently in use. The purpose of the present study was threefold. First, we aimed to investigate the different methods for CCM analysis in patients with and without small fiber neuropathy (SFN). Second, to determine the correlation of different methods for measuring corneal nerve fiber length (CNFL) and nerve fiber area (NFA). Finally, we investigated the added value of automatic NFA analysis. METHODS: We included 20 healthy controls and 80 patients with sarcoidosis, 31 with established SFN and 49 without SFN. The CNFL was measured using CCMetrics, ACCMetrics, and NeuronJ. NFA was measured with NFA FIJI and ACCMetrics NFA. RESULTS: CNFL and NFA could not distinguish sarcoidosis with and without SFN or healthy controls. CCMetrics, NeuronJ, and ACCMetrics CNFL highly correlated. Also, NFA FIJI and ACCMetrics NFA highly correlated. Reproducing a nonlinear formula between CNFL and NFA confirmed the quadratic relation between NFA FIJI and ACCMetrics CNFL. CCMetrics and NeuronJ instead showed a square root relationship and seem to be less comparable owing to differences between automatic and manual techniques. CONCLUSIONS: ACCMetrics can be used for fully automatic analysis of CCM images to optimize efficiency. However, CNFL and NFA do not seem to have a discriminatory value for SFN in sarcoidosis. Further research is needed to determine the added value and normative values of NFA in CCM analysis. TRANSLATIONAL RELEVANCE: Our study improves the knowledge about CCM software and pathophysiology of SFN

New phenotyping questionnaire for diagnosing sarcoidosis-Associated small fiber neuropathy

Small fiber neuropathy is a common complication in patients with sarcoidosis and its prevalence is estimated at 40-86%. The underlying mechanism influences the presentation of small fiber neuropathy. For example, patients with metabolic diseases are often associated with a classic length-dependent small fiber neuropathy pattern, while patients with inflammatory diseases are more often present with a non-length-dependent small fiber neuropathy. Detailed phenotyping may be useful to improve diagnostic efficiency, as a clue to underlying mechanisms and as a precondition for personalized medicine. This study examined four phenotypes distinguishing between length-dependent and non-length-dependent presentation with a new subdivision for continuous and intermittent presentation. Forty-eight sarcoid patients with symptoms and at least two clinical signs of small fiber neuropathy and normal nerve conduction studies were classified as having probable small fiber neuropathy. A new small fiber neuropathy phenotyping questionnaire has been developed that allows patients to mark the anatomical locations of pain at three different levels: The skin, muscles, and joints. The location of symptoms was used to define length dependence, and two colors were used to distinguish continuous (red) from intermittent (blue) symptoms. In addition, skin biopsy, corneal confocal microscopy, Sudoscan and water immersion skin wrinkling were used to investigate a correlation between the four phenotypes, sensory function, nerve fiber density, and autonomic nerve function. Overall, 35% of patients with probable small fiber neuropathy showed length-dependent symptoms and 44% showed non-length-dependent symptoms while 21% suffered from non-neuropathic musculoskeletal pain. The distinction between intermittent and continuous symptoms showed significantly less continuous than intermittent non-length-dependent symptoms (odds ratio = 0.3, P = 0.01). Moreover, continuous length-dependent symptoms were the only phenotype that correlated with thermal threshold testing (R = 0.3; P = 0.02) and the small fiber neuropathy screening list (R = 0.3; P = 0.03). In addition, thermal threshold testing (TTT) also correlated with the small fiber neuropathy (SFN) screening list (R = 0.3; P = 0.03). Other diagnostic methods showed no correlation with any of the four defined phenotypes. A novel finding is that TTT is only associated with continuous length-dependent pain, suggesting that TTT could result in more false negatives in patients with other pain phenotypes. Determining the pathophysiologic mechanisms could help develop new diagnostic methods. If patients suspected of SFN show symptoms without a length-dependent continuous presentation, the diagnosis should focus less on the diagnostic methods used

De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology

Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains