Coverage Of Region Research Articles

The health benefits of greening strategies to cool urban environments – A heat health impact method

Green infrastructure has the potential to cool urban environments and reduce the health burden due to heatwaves. This study develops a new method to quantify the benefits of urban heat mitigation technologies on human heat balance and population mortality. The Heat Health Impact (HHI) method is based on the state-of-the-art, multi-parameter model, Universal Thermal Climate Index (UTCI).A proof-of-concept exercise applied the HHI method to Sydney, Australia (population = 5.7 million). All available weather stations (10) were selected for full spatial coverage of the Sydney region (12,367 km2), and average hourly UTCI was calculated from meteorological observations spanning the entire year 2017. In the baseline analysis, average daily UTCI values were calculated for each of the 10 observation sites, and then spatially interpolated across the entire Sydney region for Feb 9, 2017, a representative heatwave day for Sydney. Three different greening intervention scenarios were investigated, and daily average change in UTCI (ΔUTCI) was calculated under each by comparison with the baseline scenario; this ΔUTCI was named Urban Cooling Effect (UCE). We implemented a health impact assessment methodology to estimate the change in attributable mortality due to each greening scenario for the Sydney GMR population for the representative heatwave day. Urban greening infrastructure scenarios reduced daily average UTCI between −0.2 and −1.7 °C on the heatwave day, with the health impact assessment indicating heat attributable deaths reducing up to 11.7 per day across the Sydney GMR compared to the baseline scenario. Our results highlight the health benefits of greening infrastructure to cool urban environments.

A Community-Based Management of COVID-19 in a Mobile Container Unit.

Vaccine uptake is a multifactor measure of successful immunization outcomes that includes access to healthcare and vaccine hesitancy for both healthcare workers and communities. The present coronavirus disease (COVID-19) pandemic has highlighted the need for novel strategies to expand vaccine coverage in underserved regions. Mobile clinics hold the promise of ameliorating such inequities, although there is a paucity of studies that validate environmental infection in such facilities. Here, we describe community-based management of COVID-19 through a Smart Pod mobile clinic deployed in an underserved community area in the United States (Aldine, Harris County, TX, USA). In particular, we validate infection control and biological decontamination of the Smart Pod by testing surfaces and the air-filtration system for the COVID-19 virus and bacterial pathogens. We show the Smart Pod to be efficacious in providing a safe clinical environment for vaccine delivery. Moreover, in the Smart Pod, up-to-date education of community healthcare workers was provided to reduce vaccine hesitancy and improve COVID-19 vaccine uptake. The proposed solution has the potential to augment existing hospital capacity and combat the COVID-19 pandemic locally and globally.

Comprehensive and accurate genetic variant identification from contaminated and low-coverage Mycobacterium tuberculosis whole genome sequencing data.

Improved understanding of the genomic variants that allow Mycobacterium tuberculosis (Mtb) to acquire drug resistance, or tolerance, and increase its virulence are important factors in controlling the current tuberculosis epidemic. Current approaches to Mtb sequencing, however, cannot reveal Mtb’s full genomic diversity due to the strict requirements of low contamination levels, high Mtb sequence coverage and elimination of complex regions. We have developed the XBS (compleX Bacterial Samples) bioinformatics pipeline, which implements joint calling and machine-learning-based variant filtering tools to specifically improve variant detection in the important Mtb samples that do not meet these criteria, such as those from unbiased sputum samples. Using novel simulated datasets, which permit exact accuracy verification, XBS was compared to the UVP and MTBseq pipelines. Accuracy statistics showed that all three pipelines performed equally well for sequence data that resemble those obtained from culture isolates of high depth of coverage and low-level contamination. In the complex genomic regions, however, XBS accurately identified 9.0 % more SNPs and 8.1 % more single nucleotide insertions and deletions than the WHO-endorsed unified analysis variant pipeline. XBS also had superior accuracy for sequence data that resemble those obtained directly from sputum samples, where depth of coverage is typically very low and contamination levels are high. XBS was the only pipeline not affected by low depth of coverage (5–10×), type of contamination and excessive contamination levels (>50 %). Simulation results were confirmed using whole genome sequencing (WGS) data from clinical samples, confirming the superior performance of XBS with a higher sensitivity (98.8%) when analysing culture isolates and identification of 13.9 % more variable sites in WGS data from sputum samples as compared to MTBseq, without evidence for false positive variants when rRNA regions were excluded. The XBS pipeline facilitates sequencing of less-than-perfect Mtb samples. These advances will benefit future clinical applications of Mtb sequencing, especially WGS directly from clinical specimens, thereby avoiding in vitro biases and making many more samples available for drug resistance and other genomic analyses. The additional genetic resolution and increased sample success rate will improve genome-wide association studies and sequence-based transmission studies.

Spatial distributions of <i>X</i><sub>CO<sub>2</sub></sub> seasonal cycle amplitude and phase over northern high-latitude regions

Abstract. Satellite-based observations of atmospheric carbon dioxide (CO2) provide measurements in remote regions, such as the biologically sensitive but undersampled northern high latitudes, and are progressing toward true global data coverage. Recent improvements in satellite retrievals of total column-averaged dry air mole fractions of CO2 (XCO2) from the NASA Orbiting Carbon Observatory 2 (OCO-2) have allowed for unprecedented data coverage of northern high-latitude regions, while maintaining acceptable accuracy and consistency relative to ground-based observations, and finally providing sufficient data in spring and autumn for analysis of satellite-observed XCO2 seasonal cycles across a majority of terrestrial northern high-latitude regions. Here, we present an analysis of XCO2 seasonal cycles calculated from OCO-2 data for temperate, boreal, and tundra regions, subdivided into 5∘ latitude by 20∘ longitude zones. We quantify the seasonal cycle amplitudes (SCAs) and the annual half drawdown day (HDD). OCO-2 SCAs are in good agreement with ground-based observations at five high-latitude sites, and OCO-2 SCAs show very close agreement with SCAs calculated for model estimates of XCO2 from the Copernicus Atmosphere Monitoring Services (CAMS) global inversion-optimized greenhouse gas flux model v19r1 and the CarbonTracker2019 model (CT2019B). Model estimates of XCO2 from the GEOS-Chem CO2 simulation version 12.7.2 with underlying biospheric fluxes from CarbonTracker2019 (GC-CT2019) yield SCAs of larger magnitude and spread over a larger range than those from CAMS, CT2019B, or OCO-2; however, GC-CT2019 SCAs still exhibit a very similar spatial distribution across northern high-latitude regions to that from CAMS, CT2019B, and OCO-2. Zones in the Asian boreal forest were found to have exceptionally large SCA and early HDD, and both OCO-2 data and model estimates yield a distinct longitudinal gradient of increasing SCA from west to east across the Eurasian continent. In northern high-latitude regions, spanning latitudes from 47 to 72∘ N, longitudinal gradients in both SCA and HDD are at least as pronounced as latitudinal gradients, suggesting a role for global atmospheric transport patterns in defining spatial distributions of XCO2 seasonality across these regions. GEOS-Chem surface contact tracers show that the largest XCO2 SCAs occur in areas with the greatest contact with land surfaces, integrated over 15–30 d. The correlation of XCO2 SCA with these land surface contact tracers is stronger than the correlation of XCO2 SCA with the SCA of CO2 fluxes or the total annual CO2 flux within each 5∘ latitude by 20∘ longitude zone. This indicates that accumulation of terrestrial CO2 flux during atmospheric transport is a major driver of regional variations in XCO2 SCA.

Resistance to Mogamulizumab Is Associated with Loss of CCR4 in Cutaneous T Cell Lymphoma

Background: Mogamulizumab is a humanized anti-CCR4 antibody approved for the treatment of mycosis fungoides and Sézary Syndrome. Despite almost universal expression of CCR4 in these diseases, many patients eventually develop resistance to mogamulizumab (moga). The mechanisms of resistance to moga have not been characterized. An understanding of the resistance mechanisms will shed light on the disease biology and inform selection of next line therapies and the development of future CCR4-directed treatments for patients with cutaneous T cell lymphoma (CTCL).Methods: We identified 19 patients with mycosis fungoides or Sezary syndrome in whom moga had been discontinued due to lack or loss of response to therapy. Archival blood and skin and/or lymph node biopsies after discontinuation of treatment were studied. Specimens collected prior to initiation of moga were also procured whenever available. CCR4 protein expression was determined by immunohistochemistry (IHC; clone L291H4; Biolegend). Targeted sequencing was performed using the Agilent XTHS2 platform (Agilent) with a custom Agilent SureSelect panel that included full coverage of the exonic regions of CCR4.Results:Fourteen patients had Sézary syndrome and five had mycosis fungoides. Twelve patients were classified as primary refractory to treatment and the remaining seven patients had secondary resistance. Median duration of treatment with moga was 3.5 months (range: 2-41). CCR4 expression was non-detectable in post-treatment specimens of 8 of 14 (57%) patients with evaluable specimens. Targeted DNA sequencing of post-treatment samples revealed novel coding mutations of CCR4 in three patients. Only one of the 3 mutations (L21V) overlapped with the known N-terminal mogamulizumab binding epitope. The other two mutations occurred in transmembrane domains (M116R, T161-W162 delinsSR). Two patients with mutations in CCR4 also had unequivocal copy number loss involving the CCR4 locus. Acquisition of CCR4 genomic alterations corresponded with lack of CCR4 expression by immunohistochemistry. In-vitro validation of the novel CCR4 mutations is ongoing and results from these experiments will be included in the final presentation.Conclusion: Overall our study indicates that potential loss of CCR4 expression, with or without underlying genomic aberrations, represents a major hurdle in the development of future CCR4 targeting strategies in CTCL. This finding has important implications for management and monitoring of cutaneous T cell lymphoma patients receiving CCR4 directed therapies. DisclosuresBeygi: Kite/Gilead: Current Employment. Kim: Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; CRISPR: Research Funding; Eisai: Research Funding; Soligenix: Research Funding; Portola: Membership on an entity's Board of Directors or advisory committees, Research Funding; Elorac: Research Funding; Galderma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trillium: Research Funding; Corvus: Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Kirin: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Secura Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion, AstraZeneca Rare Disease: Other: Study investigator; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Horizon: Research Funding. Khodadoust: Alexion, AstraZeneca Rare Disease: Other: Study investigator; Myeloid Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics, Nutcracker Therapeutics: Research Funding.

Analysis of Whistler‐Mode and Z‐Mode Emission in the Juno Primary Mission

AbstractAt the end of the Juno primary mission, we report observations of whistler mode chorus and Z‐mode emission. The Juno orbits are evolving and much better coverage of the whistler mode chorus source region has resulted since the earlier surveys. Bursty chorus emission extending to ∼30° latitude and to frequencies less than the lower hybrid frequency near the source region imply high electron energies (>100 keV). Average chorus intensity levels peak at ∼10−3 nT2 near M‐shell of 8–9 and magnetic latitude of ∼5°. Z‐mode emission is identified at higher latitudes generally near and inward of the Io torus with intensity levels as much as two orders of magnitude higher than Z‐mode emissions observed at Saturn. Inferred source regions for the Z‐mode are consistent with the inner edge of the Io torus and with auroral field lines that may also support Jovian kilometric and decametric emission. Parametric fitting functions are evaluated for both whistler mode chorus and Z‐mode, describing wave intensity as a function of frequency, magnetic latitude, and M‐shell. Both whistler mode and Z‐mode waves may have significant impact on electron scattering and acceleration at Jupiter as recent models indicate.

Nigeria’s Polio Elimination Playbook: Lessons To Strengthening Health Systems For Other Eradicable Diseases

According to the World Health Organization (WHO), poliomyelitis (polio) is a highly infectious viral disease that largely affects children under 5 years of age. In August 2020, Nigeria was declared free of the wild poliovirus by the WHO and United Nations International Children’s Emergency Fund (UNICEF). This review assesses the available evidence on issues with Nigeria’s efforts and challenges towards the fight against poliomyelitis such as the wild poliovirus (WPV) and vaccine-derived poliovirus (VDPV). Published grey literature was assessed to make this mini-review. No restriction on publication dates was applied during the literature search. Available data showed efforts such as strong government accountability framework; introduction of geographic information systems to ensure more efficient utilization of resources and coverage; the digitalization of vaccine delivery; optimal utilization of disease notification officers (DNOs) at every unit of the local government; commitment of resources from the federal government to states that were prone to the reoccurrence of Polio; improved social mobilization, supervision, and monitoring; as well as assistance from international organizations were deplored. Some of the notable challenges faced with the eradication of polio include the refusal of parents to consent to vaccinations for their children, reoccurrences due to poor supplies of potable water, religious bias from the northern region, differential routines, and supplemental immunization coverage. The lessons learned in polio eradication can be channeled into the elimination of other diseases, but efforts must be in place to continue to monitor the reoccurrence of polio and other emerging diseases through surveillance systems.

Validation of amplicon-based next generation sequencing panel for second-tier test in newborn screening for inborn errors of metabolism

Abstract Objectives Next generation sequencing (NGS) technology has allowed cost-effective massive parallel DNA sequencing. To evaluate the utility of NGS for newborn screening (NBS) of inborn errors of metabolism (IEM), a custom panel was designed to target 87 disease-related genes. The pilot study was primarily proposed for second-tier testing under the NBSIEM program in Hong Kong. Methods The validation of the panel was performed with two reference genomes and an external quality assurance (EQA) sample. Sequencing libraries were synthesized with amplicon-based approach. The libraries were pooled, spiked-in with 2% PhiX DNA as technical control, for 16-plex sequencing runs. Sequenced reads were analyzed using a commercially available pipeline. Results The average target region coverage was 208× and the fraction of region with target depth ≥20× was 95.7%, with a sensitivity of 91.2%. There were 85 out of 87 genes with acceptable coverage, and EQA result was satisfactory. The turnaround time from DNA extraction to completion of variant calling and quality control (QC) procedures was 2.5 days. Conclusions The NGS approach with the amplicon-based panel has been validated for analytical performance and is suitable for second-tier NBSIEM test.

Multiple Region Coverage Path Planning for Autonomous Underwater Vehicle.

Coverage path planning methodology for an autonomous underwater vehicle to search multiple non-overlapping regions has been proposed in the paper. The proposed methodology is based on the genetic algorithm (GA). The GA used in the proposed methodology has been tuned for the specific problem, using design of experiment on an equivalent travelling salesman problem benchmark instance. Optimality of the generated paths was analysed through simulation studies. Results indicated that the proposed methodology generated shorter paths in comparison to conventional methods.

Protest Event Analysis Under Conditions of Limited Press Freedom: Comparing Data Sources

The investigation of long-term trends in contentious politics relies heavily on protest event analysis based on newspaper reports. This tends to be problematic in restricted media environments. To mitigate the effects of bias and (self-)censorship, researchers of protest in authoritarian regimes have experimented with other sources such as international media and dissident websites. However, even though classical news media are easier targets for repression, journalistic reports might still outperform other sources regarding the quality of information provided. Although these advantages and disadvantages are known in the literature, different types of sources have seldom been tested against each other in an authoritarian context. Using the example of Russia between 2007 and 2012, the present article systematically compares protest event data from English-language news agencies, dissident websites, and several local sources, first and foremost with a view to improving methodological knowledge. The analysis addresses broad trends across time and space as well as the coverage of specific regions and single protest events. It finds that although the data sources paint different pictures of protest in Russia, this divergence is systematic and can be put to productive use. The article closes with a discussion on how its findings can be applied in other contexts.

P59.02 Profile of Next-Generation Sequencing (NGS) on MET exon 14 Skipping Mutation and MET Amplification in Lung Cancer: A Calibration Project in China

MET is an oncogenic driver and becomes a significant therapeutic target in lung cancer. Detection of MET alterations is essential for clinical treatment decision-making. With the advancement of diagnostic technology, next-generation sequencing (NGS) becomes an option. However, it has limited probe design and coverage of specific regions of introns, and the quality of testing varies widely among different laboratories. Moreover, clinically defined cut-off values for MET amplification vary considerably and lack uniform criteria.

Geometry and dynamics of passive scalar structures in compressible turbulent mixing

We present a structure-based numerical analysis of passive scalar mixing in decaying homogeneous isotropic turbulence (DHIT) and shock-turbulence interaction canonical configurations. The analysis focuses on the temporal evolution of ensembles of passive scalar structures, initialized as spheres of different sizes relative to the Taylor microscale. An algorithm is introduced to track the evolution of each individual structure and the interactions with other structures in the ensemble, relating changes in the surface geometry and the underlying physical processes (turbulent transport, scalar dissipation, and shock compression). The tracking algorithm is applied to datasets from shock-capturing direct numerical simulations of DHIT, with Taylor microscale Reynolds number Reλ=40 and turbulence Mach number Mt=0.2, and STI cases in which the turbulence is processed by a shock wave at Mach numbers M = 1.5 and 3.0. Temporal surface convolution increases for initially larger structures, resulting in a higher probability of locally hyperbolic geometries where breakup into smaller structures occurs. Shock-induced deformation of the structures amplifies breakup processes, enhancing mixing, particularly for larger structures. Mixing enhancement by the shock is manifested as an amplification of the surface-averaged scalar gradient, which increases for initially larger structures. The alignment between the scalar gradient and the most extensional strain-rate eigendirection on the scalar isosurfaces also increases across the shock. Larger magnitudes of the scalar gradient and its alignment with the most compressive strain-rate eigendirection correlate with flatter surface regions. Shock-induced structure compression increases the area coverage of flat regions, where the amplification of scalar gradient is localized.

Japanese encephalitis in children from an endemic district of West Bengal, India: A hospital based observational study

Background: Japanese encephalitis (JE) is an important cause of viral encephalitis in children in South East Asian countries including India. Aims and Objective: We have done this study in an JE endemic district of India to know the demographic profile, clinical presentation, seasonal variation, outcome and about the vaccination status of the JE cases. Materials and Methods: This observational, cross sectional study was done for a period of one year in the Pediatric department of Burdwan Medical College, West Bengal in children up to the age of 12 year, presented with acute encephalitis syndrome (AES). Demographic data, clinical presentation and JE vaccination status were recorded and confirmed JE cases were detected by either serum or CSF JE IgM antibody. Outcome of the JE cases were noted as discharge, death or leave against medical advice (LAMA). Results: We have found confirmed JE in 18 children out of total 125 AES cases (14.4%). 61.1% of them were within 6 to 12 year age, mostly from low socioeconomic status and 100% case occurred during monsoon and post monsoon period. History, was of JE vaccination were present in only 11.1% case of JE. Most common presenting symptoms were high fever (100%), convulsion (94.4%) and altered sensorium (72.2%). 77.8% JE case were discharged and 11.1% expired. Conclusion: We must consider JE whenever a child from any endemic district comes with AES and we should try to bring every child under the JE vaccination coverage in the endemic regions globally to reduce the burden of this preventable encephalitis.

Monitoring Changes to Arctic Vegetation and Glaciers at Ny-Ålesund, Svalbard, Based on Time Series Remote Sensing

Climate change has profoundly affected global ecological security. The most vulnerable region on Earth is the high-latitude Arctic. Identifying the changes in vegetation coverage and glaciers in high-latitude Arctic coastal regions is important for understanding the process and impact of global climate change. Ny-Ålesund, the northern-most human settlement, is typical of these coastal regions and was used as a study site. Vegetation and glacier changes over the past 35 years were studied using time series remote sensing data from Landsat 5/7/8 acquired in 1985, 1989, 2000, 2011, 2015 and 2019. Site survey data in 2019, a digital elevation model from 2009 and meteorological data observed from 1985 to 2019 were also used. The vegetation in the Ny-Ålesund coastal zone showed a trend of declining and then increasing, with a breaking point in 2000. However, the area of vegetation with coverage greater than 30% increased over the whole study period, and the wetland moss area also increased, which may be caused by the accelerated melting of glaciers. Human activities were responsible for the decline in vegetation cover around Ny-Ålesund owing to the construction of the town and airport. Even in areas with vegetation coverage of only 13%, there were at least five species of high-latitude plants. The melting rate of five major glaciers in the study area accelerated, and approximately 82% of the reduction in glacier area occurred after 2000. The elevation of the lowest boundary of the five glaciers increased by 50–70 m. The increase in precipitation and the average annual temperature after 2000 explains the changes in both vegetation coverage and glaciers in the study period.

A Long-Term Ecological Vulnerability Analysis of the Tibetan Region of Natural Conditions and Ecological Protection Programs

The combined impacts of drastic natural environment change and increasing human interference are making the uncertainty of the Tibetan Plateau’s ecological vulnerability the world’s largest. In this study, an ecological vulnerability index (EVI) of Tibet in the core area of the Tibetan Plateau was assessed using a selected set of ecological, social, and economic indicators and using a spatial principal component analysis (SPCA) to calculate their weights. The data included Landsat images and socio-economic data from 1990 to 2015 in five-year intervals. The results showed that the total EVI remained at a high vulnerability level, with drastic fluctuation from 1990 to 2000 (a peak in 1995, when there was a sudden increase in light vulnerability, which moved to extreme vulnerability in the next period), and minor fluctuations after 2000, gradually increasing from southeast to northwest. In addition, the spatial analysis showed a distinct positive correlation between the EVI and grassland area (0.33), land use degree (0.15), NDVI (0.14), livestock husbandry output, and a negative correlation in terms of desertification area. The artificial afforestation program (AAP) had a positive significant correlation with NDVI (R2 = 0.88), preventing the environment from becoming more vulnerable. The results provide practical information and suggestions for planners to improve the land use degree in urban areas and the vegetation coverage in pastoral regions of the Tibetan Plateau based on the spatial–temporal heterogeneity patterns of the EVI of Tibet.

Presentation of a Model for the Distribution of Emergency First Aid During Pre-hospital Triage with a Practical Example

Background: The proper triage of patients, supply chain management, and proper location, delivery, and distribution of health facilities and services in times of crises and disasters have always been challenging issues playing an important role in reducing damages and increasing the level of patient care during emergencies. Objectives: This study was conducted to design a model for allocating emergency first aid in pre-hospital triage. Methods: The model presented in this study is a two-level mathematical model using nonlinear programming to introduce a new location for the establishment and activation of distribution centers in the supply chain and distribution of pre-hospital health-oriented services during crises. This proposed hybrid model seeks to strike a balance between the concepts of efficiency, cost, and dispersion. By expanding the insurance coverage of health services, the number of services provided in the times of crises and triage of injured patients also increases, leading to the delivery of more profits and improvement in the effectiveness of medical services in the shortest possible time. Results: This model helps to select suitable candidate locations for the triage of patients and increases its performance. Therefore, the appropriateness of each place is measured according to the criteria set for them. This model considers different parameters for selecting places for the distribution and allocation of emergency services, including the efficiency of the centers selected for the triage of patients, the cost of the inventory system for providing primary services, the dispersion of selected triage centers, demands for sick and injured patients, the possibility of constructing additional distribution centers (e.g., field hospitals), and strategies to avoid the imposition of additional costs. Conclusions: This study showed that by maximizing the dispersion of desirable facilities, such as distribution centers, the demand coverage of different regions is appropriately improved. In other words, the health service distributor, as one of the important parts of the supply chain, helps service providers better meet the needs of injured patients during emergencies.

The Role of Correlation in the Doubly Dirty Fading MAC With Side Information at the Transmitters

We investigate the impact of fading correlation on the performance of the doubly dirty fading multiple access channel (MAC) with non-causally known side information at transmitters. Using Copula theory, we derive closed-form expressions for the outage probability (OP) and the coverage region under positive/negative dependence conditions. We show that a positive dependence structure between the fading channel coefficients is beneficial for the system performance, as it improves the OP and extends the coverage region compared to the case of independent fading. Conversely, a negative dependence structure has a detrimental effect on both performance metrics.

Detecting copy number variation in next generation sequencing data from diagnostic gene panels

BackgroundDetection of copy number variation (CNV) in genes associated with disease is important in genetic diagnostics, and next generation sequencing (NGS) technology provides data that can be used for CNV detection. However, CNV detection based on NGS data is in general not often used in diagnostic labs as the data analysis is challenging, especially with data from targeted gene panels. Wet lab methods like MLPA (MRC Holland) are widely used, but are expensive, time consuming and have gene-specific limitations. Our aim has been to develop a bioinformatic tool for CNV detection from NGS data in medical genetic diagnostic samples.ResultsOur computational pipeline for detection of CNVs in NGS data from targeted gene panels utilizes coverage depth of the captured regions and calculates a copy number ratio score for each region. This is computed by comparing the mean coverage of the sample with the mean coverage of the same region in other samples, defined as a pool. The pipeline selects pools for comparison dynamically from previously sequenced samples, using the pool with an average coverage depth that is nearest to the one of the samples. A sliding window-based approach is used to analyze each region, where length of sliding window and sliding distance can be chosen dynamically to increase or decrease the resolution. This helps in detecting CNVs in small or partial exons. With this pipeline we have correctly identified the CNVs in 36 positive control samples, with sensitivity of 100% and specificity of 91%. We have detected whole gene level deletion/duplication, single/multi exonic level deletion/duplication, partial exonic deletion and mosaic deletion. Since its implementation in mid-2018 it has proven its diagnostic value with more than 45 CNV findings in routine tests.ConclusionsWith this pipeline as part of our diagnostic practices it is now possible to detect partial, single or multi-exonic, and intragenic CNVs in all genes in our target panel. This has helped our diagnostic lab to expand the portfolio of genes where we offer CNV detection, which previously was limited by the availability of MLPA kits.

Development and validation of a flexible DNA extraction (PAN) method for liquid biopsy of multiple sample types.

BackgroundLiquid biopsy is gaining increasing popularity in cancer screening and diagnosis. However, there is no relatively mature DNA isolation method or commercial kit available that is compatible with different LB sample types. This study developed a PAN‐sample DNA isolation method (PAN method) for liquid biopsy samples.MethodsThe PAN method has two key steps, including biosample‐specific pretreatments for various LB sample types and high concentration guanidine thiocyanate buffer for lysis and denaturation procedure. Subsequently, the performance of PAN method was validated by a series of molecular analyses.ResultsThe PAN method was used to isolate DNA from multiple sample types related to LB, including plasma, serum, saliva, nasopharyngeal swab, and stool. All purified DNA products showed good quality and high quantity. Comparison of KRAS mutation analysis using DNA purified using PAN method versus QIAamp methods showed similar efficiency. Epstein‐Barr virus DNA was detected via Q‐PCR using DNA purified from serum, plasma, nasopharyngeal swab, and saliva samples collected from nasopharyngeal carcinoma patients. Similarly, methylation sequencing of swab and saliva samples revealed good coverage of target region and high methylation of HLA‐DPB1 gene. Finally, 16S rDNA gene sequencing of saliva, swab, and stool samples successfully defines the relative abundance of microbial communities.ConclusionsThis study developed and validated a PAN‐sample DNA isolation method that can be used for different LB samples, which can be applied to molecular epidemiological research and other areas.

RESCOT: Restriction enzyme set and combination optimization tools for rNMP capture techniques

The incorporation of ribonucleoside monophosphates (rNMPs) in genomic DNA is a frequent phenomenon in many species, often associated with genome instability and disease. The ribose-seq technique is one of a few techniques designed to capture and map rNMPs embedded in genomic DNA. The first step of ribose-seq is restriction enzyme (RE) fragmentation, which cuts the genome into smaller fragments for subsequent rNMP capture. The RE selection chosen for genomic DNA fragmentation in the first step of the rNMP-capture techniques determines the genomic regions in which the rNMPs can be captured. Here, we designed a computational method, Restriction Enzyme Set and Combination Optimization Tools (RESCOT), to calculate the genomic coverage of rNMP-captured regions for a given RE set and to optimize the RE set to significantly increase the rNMP-captured-region coverage. Analyses of ribose-seq libraries for which the RESCOT tools were applied reveal that many rNMPs were captured in the expected genomic regions. Since different rNMP-mapping techniques utilize RE fragmentation and purification steps based on size-selection of the DNA fragments in the protocol, we discuss the possible usage of RESCOT for other rNMP-mapping techniques. In summary, RESCOT generates optimized RE sets for the fragmentation step of many rNMP capture techniques to maximize rNMP capture rate and thus enable researchers to better study characteristics of rNMP incorporation.